Aminoglycosides and uses thereof in treating genetic disorders

ABSTRACT

A new class of pseudo-trisaccharide aminoglycosides having an alkyl group at the 5″ position, exhibiting efficient stop codon mutation readthrough activity, low cytotoxicity and high selectivity towards eukaryotic translation systems are provided. Also provided are pharmaceutical compositions containing the same, and uses thereof in the treatment of genetic disorders, as well as processes of preparing these aminoglycosides. The disclosed aminoglycosides can be represented by the general formula I: 
                         
or a pharmaceutically acceptable salt thereof, wherein R 1  is selected from the group consisting of alkyl, cycloalkyl and aryl; and all other variables and features are as described in the specification.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/914,045 filed on Mar. 7, 2018, which is a continuation of U.S. patentapplication Ser. No. 15/453,990 filed on Mar. 9, 2017, which is adivision of U.S. patent application Ser. No. 14/866,960 filed on Sep.27, 2015, now U.S. Pat. No. 9,616,079, which is a continuation of U.S.patent application Ser. No. 14/461,477 filed on Aug. 18, 2014, now U.S.Pat. No. 9,175,029, which is a continuation of U.S. patent applicationSer. No. 13/885,715 filed on May 16, 2013, now U.S. Pat. No. 8,895,519,which is a National Phase of PCT Patent Application No.PCT/IL2011/000889 having International Filing Date of Nov. 17, 2011,which claims the benefit of priority of U.S. Provisional PatentApplication No. 61/414,956 filed on Nov. 18, 2010. The contents of theabove applications are all incorporated by reference as if fully setforth herein in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Contract No.GM094792 awarded by the NIH. The government has certain rights in theinvention.

SEQUENCE LISTING STATEMENT

The ASCII file, entitled 2019-11-25 Sequence-Listing Text-File125870-8002.US06, created and filed on Nov. 25, 2019, comprising 6 KB,is incorporated herein by reference.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to a newclass of aminoglycosides and more particularly, but not exclusively, tonovel aminoglycosides with improved efficacy towards treatment ofgenetic disorders.

Many human genetic disorders result from nonsense mutations, where oneof the three stop codons (UAA, UAG or UGA) replaces an amino acid-codingcodon, leading to premature termination of the translation andeventually to truncated inactive proteins. Currently, hundreds of suchnonsense mutations are known, and several were shown to account forcertain cases of fatal diseases, including cystic fibrosis (CF),Duchenne muscular dystrophy (DMD), ataxia-telangiectasia, Hurlersyndrome, hemophilia A, hemophilia B, Tay-Sachs, and more. For many ofthose diseases there is presently no effective treatment, and althoughgene therapy seems like a potential possible solution for geneticdisorders, there are still many critical difficulties to be solvedbefore this technique could be used in humans.

Certain aminoglycosides have been shown to have therapeutic value in thetreatment of several genetic diseases because of their ability to induceribosomes to read-through stop codon mutations, generating full-lengthproteins from part of the mRNA molecules.

Typically, aminoglycosides are highly potent, broad-spectrum antibioticscommonly used for the treatment of life-threatening infections. It isaccepted that the mechanism of action of aminoglycoside antibiotics,such as paromomycin, involves interaction with the prokaryotic ribosome,and more specifically involved binding to the decoding A-site of the 16Sribosomal RNA, which leads to protein translation inhibition andinterference with the translational fidelity.

Several achievements in bacterial ribosome structure determination,along with crystal and NMR structures of bacterial A-siteoligonucleotide models, have provided useful information forunderstanding the decoding mechanism in prokaryote cells andunderstanding how aminoglycosides exert their deleterious misreading ofthe genetic code. These studies and others have given rise to thehypothesis that the affinity of the A-site for a non-cognate mRNA-tRNAcomplex is increased upon aminoglycosides binding, preventing theribosome from efficiently discriminating between non-cognate and cognatecomplexes.

The enhancement of termination suppression by aminoglycosides ineukaryotes is thought to occur in a similar mechanism to theaminoglycosides' activity in prokaryotes of interfering withtranslational fidelity during protein synthesis, namely the binding ofcertain aminoglycosides to the ribosomal A-site probably induceconformational changes that stabilize near-cognate mRNA-tRNA complexes,instead of inserting the release factor. Aminoglycosides have been shownto suppress various stop codons with notably different efficiencies(UGA>UAG>UAA), and the suppression effectiveness is further dependentupon the identity of the fourth nucleotide immediately downstream fromthe stop codon (C>U>A≥grams) as well as the local sequence contextaround the stop codon.

The desired characteristics of an effective read-through drug would beoral administration and little or no effect on bacteria. Antimicrobialactivity of read-through drug is undesirable as any unnecessary use ofantibiotics, particularly with respect to the gastrointestinal (GI)biota, due to the adverse effects caused by upsetting the GI biotaequilibrium and the emergence of resistance. In this respect, inaddition to the abovementioned limitations, the majority of clinicalaminoglycosides are greatly selective against bacterial ribosomes, anddo not exert a significant effect on cytoplasmic ribosomes of humancells.

In an effort to circumvent the abovementioned limitations, thebiopharmaceutical industry is seeking new stop mutations suppressiondrugs by screening large chemical libraries for nonsense read-throughactivity. Using this approach, a non-aminoglycoside compound,3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid (PTC124), hasbeen discovered. The facts that PTC124 is reported to have noantibacterial activity and no reported toxicity, suggest that itsmechanism of action on the ribosome is different than that of theaminoglycosides.

The fact that aminoglycosides could suppress premature nonsensemutations in mammalian cells was first demonstrated by Burke and Mogg in1985, who also noted the therapeutic potential of these drugs in thetreatment of genetic disorders. The first genetic disease examined wascystic fibrosis (CF), the most prevalent autosomal recessive disorder inthe Caucasian population, affecting 1 in 2,500 newborns. CF is caused bymutations in the cystic fibrosis transmembrane conductance regulator(CFTR) protein. Currently, more than 1,000 different CF-causingmutations in the CFTR gene were identified, and 5-10% of the mutationsare premature stop codons. In Ashkenazi Jews, the W1282X mutation andother nonsense mutations account for 64% of all CFTR mutant alleles.

The first experiments of aminoglycoside-mediated suppression of CFTRstop mutations demonstrated that premature stop mutations found in theCFTR gene could be suppressed by members of the gentamicin family andgeniticin (G-418), as measured by the appearance of full-length,functional CFTR in bronchial epithelial cell lines.

Suppression experiments of intestinal tissues from CFTR−/− transgenicmice mutants carrying a human CFTR-G542X transgene showed that treatmentwith gentamicin, and to lesser extent tobramycin, have resulted in theappearance of human CFTR protein at the glands of treated mice. Mostimportantly, clinical studies using double-blind, placebo-controlled,crossover trails have shown that gentamicin can suppress stop mutationsin affected patients, and that gentamicin treatment improvedtransmembrane conductance across the nasal mucosa in a group of 19patients carrying CFTR stop mutations. Other genetic disorders for whichthe therapeutic potential of aminoglycosides was tested in in-vitrosystems, cultured cell lines, or animal models include DMD, Hurlersyndrome, nephrogenic diabetes insipidus, nephropathic cystinosis,retinitis pigmentosa, and ataxia-telangiectasia.

However, one of the major limitations in using aminoglycosides aspharmaceuticals is their high toxicity towards mammals, typicallyexpressed in kidney (nephrotoxicity) and ear-associated (ototoxicity)illnesses. The origin of this toxicity is assumed to result from acombination of different factors and mechanisms such as interactionswith phospholipids, inhibition of phospholipases and the formation offree radicals. Although considered selective to bacterial ribosomes,most aminoglycosides bind also to the eukaryotic A-site but with loweraffinities than to the bacterial A-site. The inhibition of translationin mammalian cells is also one of the possible causes for the hightoxicity of these agents. Another factor adding to their cytotoxicity istheir binding to the mitochondrial ribosome at the 12S rRNA A-site,whose sequence is very close to the bacterial A-site.

Many studies have been attempted to understand and offer ways toalleviate the toxicity associated with aminoglycosides, including theuse of antioxidants to reduce free radical levels, as well as the use ofpoly-L-aspartate and daptomycin, to reduce the ability ofaminoglycosides to interact with phospholipids. The role of megalin (amultiligand endocytic receptor which is especially abundant in thekidney proximal tubules and the inner ear) in the uptake ofaminoglycosides has recently been demonstrated. The administration ofagonists that compete for aminoglycoside binding to megalin alsoresulted in a reduction in aminoglycoside uptake and toxicity. Inaddition, altering the administration schedule and/or the manner inwhich aminoglycosides are administered has been investigated as means toreduce toxicitys.

Despite extensive efforts to reduce aminoglycoside toxicity, few resultshave matured into standard clinical practices and procedures for theadministration of aminoglycosides to suppress stop mutations, other thanchanges in the administration schedule. For example, the use ofsub-toxic doses of gentamicin in the clinical trails probably caused thereduced read-through efficiency obtained in the in-vivo experimentscompared to the in-vitro systems. The aminoglycoside geneticine (G-418sulfate) showed the best termination suppression activity in in-vitrotranslation-transcription systems, however, its use as a therapeuticagent is not possible since it is lethal even at very lowconcentrations. For example, the LD₅₀ of G-418 against human fibroblastcells is 0.04 mg/ml, compared to 2.5-5.0 mg/ml for gentamicin, neomycinand kanamycin.

The increased sensitivity of eukaryotic ribosomes to some aminoglycosidedrugs, such as G-418 and gentamicin, is intriguing but up to date couldnot be rationally explained because of the lack of sufficient structuraldata on their interaction with eukaryotic ribosomes. Since G-418 isextremely toxic even at very low concentrations, presently gentamicin isthe only aminoglycoside tested in various animal models and clinicaltrials. Although some studies have shown that due to their relativelylower toxicity in cultured cells, amikacin and paromomycin can representalternatives to gentamicin for stop mutation suppression therapy, noclinical trials with these aminoglycosides have been reported yet.

To date, nearly all suppression experiments have been performed withclinical, commercially available aminoglycosides, however, only alimited number of aminoglycosides, including gentamicin, amikacin, andtobramycin, are in clinical use as antibiotics for internaladministration in humans. Among these, tobramycin do not have stopmutations suppression activity, and gentamicin is the onlyaminoglycoside tested for stop mutations suppression activity in animalmodels and clinical trials. Recently, a set of neamine derivatives wereshown to promote read-through of the SMN protein in fibroblasts derivedfrom spinal muscular atrophy (SPA) patients; however, these compoundswere originally designed as antibiotics and no conclusions were derivedfor further improvement of the read-through activity of thesederivatives.

WO 2007/113841, by some of the present inventors, which is incorporatedby reference as if fully set forth herein, teaches a class ofparomomycin-derived aminoglycosides, which were designed specifically toexhibit high premature stop-codon mutations readthrough activity whileexerting low cytotoxicity in mammalian cells and low antimicrobialactivity, and can thus be used in the treatment of genetic diseases.This class of paromomycin-derived aminoglycosides was designed byintroducing certain manipulations of a paromamine core, which lead toenhanced readthrough activity and reduced toxicity and antimicrobialactivity. The manipulations were made on several positions of theparomamine core.

One such manipulation or tne paromamine core wnicn nas been described inWO 2007/113841 is the determination of the beneficial role of a hydroxylgroup at position 6′ of the aminoglycoside core (see, for example, NB30and NB54 below).

Another manipulation of the paromamine core which has been defined anddemonstrated in WO 2007/113841 is the introduction of one or moremonosaccharide moieties or an oligosaccharide moiety at position 3′, 5and/or 6 of the aminoglycoside core. This manipulation is reflected as“Ring III” in the exemplary compounds NB30 and NB54 shown hereinabove.

An additional manipulation of the paromamine core which has been definedand demonstrated in WO 2007/113841 is the introduction of an(S)-4-amino-2-hydroxybutyryl (AHB) moiety at position 1 of theparomamine core. This manipulation is reflected in exemplary compoundNB54 shown hereinabove. It has been demonstrated that such anintroduction of an AHB moiety provides for enhanced readthrough activityand reduced toxicity.

An additional manipulation of the paromamine core which has beendescribed in WO 2007/113841 is the substitution of hydrogen at position6′ by an alkyl such as a methyl substituent. This manipulation has beenexemplified in a derivative of compounds NB30 and NB54, referred to asNB74 and NB84 respectively.

Additional background art includes Nudelman, I., et al., Bioorg. Med.Chem. Lett., 2006. 16(24): p. 6310-5; Hobbie, S. N., et al., NucleicAcids Res., 2007. 35(18): p. 6086-93; Kondo, J., et al., Chembiochem.,2007. 8(14): p. 1700-9; Rebibo-Sabbah, A., et al., Hum Genet, 2007.122(3-4): p. 373-81; Azimov, R., et al., Am. J. Physiol. Renal.Physiol., 2008. 295(3): p. F633-41; Hainrichson, M., et al., Org.Biomol. Chem., 2008. 6(2): p. 227-39; Hobbie, S. N., et. al., Proc.Natl. Acad. Sci. USA, 2008. 105(52): p. 20888-93; Hobbie, S. N., et.al., Proc. Natl. Acad. Sci. USA, 2008. 105(9): p. 3244-9; Nudelman, I.,et al., Adv. Synth. Catal., 2008. 350: p. 1682-1688; Nudelman, I., etal., J. Med. Chem., 2009. 52(9): p. 2836-45; Venkataraman, N., et al.,PLoS Biol., 2009. 7(4): p. e95; Brendel, C., et al., J. Mol. Med.(Berl), 2010. 89(4): p. 389-98; Goldmann, T., et al., Invest OphthalmolVis Sci, 2010. 51(12): p. 6671-80; Malik, V., et al., Ther. Adv. Neurol.Disord., 2010. 3(6): p. 379-89; Nudelman, I., et al., Bioorg. Med.Chem., 2010. 18(11): p. 3735-46; Warchol, M. E., Curr. Opin.Otolaryngol. Head Neck Surg., 2010. 18(5): p. 454-8; Lopez-Novoa, J. M.,et al., Kidney Int., 2011. 79(1): p. 33-45; Rowe, S. M., et al., J. Mol.Med. (Berl), 2011. 89(11): p. 1149-61; and Vecsler, M., et al., PLoSOne, 2011. 6(6): p. e20733.

SUMMARY OF THE INVENTION

The present invention relates to a new class of pseudo-trisaccharideaminoglycosides, which can be beneficially used in the treatment ofgenetic diseases, such as cystic fibrosis, by exhibiting high prematurestop-codon mutations read-through activity while exerting low toxicityin mammalian cells and low antimicrobial activity. The presentlydisclosed aminoglycosides are characterized by a core structure based onRings I, II and III of paromomycin with the addition of an alkyl inposition 5″ on Ring III.

Thus, according to an aspect of some embodiments of the presentinvention there is provided a compound having the general formula I:

or a pharmaceutically acceptable salt thereof, wherein:

R₁ is selected from the group consisting of alkyl, cycloalkyl and aryl;

R₂ is hydrogen or (S)-4-amino-2-hydroxybutyryl (AHB);

R₃ is selected from the group consisting of hydrogen, alkyl, cycloalkyland aryl; and

a stereo-configuration of each of position 6′ and position 5″ isindependently an R configuration or an S configuration.

According to some embodiments of the invention, R₁ is alkyl.

According to some embodiments of the invention, the alkyl is methyl.

According to some embodiments of the invention, R₂ and R₃ are eachhydrogen.

According to some embodiments of the invention, R₂ is AHB and R₃ ishydrogen.

According to some embodiments of the invention, R₂ is hydrogen and R₃ isalkyl.

According to some embodiments of the invention, R₂ is AHB and R₃ isalkyl.

According to some embodiments of the invention, the alkyl is methyl.

According to some embodiments of the invention, the compounds presentedherein are selected from the group consisting of the compounds NB118,NB119, NB122, NB123, NB124, NB125, NB127 and NB128.

According to some embodiments of the invention, the compounds presentedherein are characterized by exhibiting a ratio of IC₅₀ translationinhibition in eukaryotes to IC₅₀ translation inhibition in prokaryoteslower than 15. According to some embodiments of the invention, the ratiois lower than 1.

According to some embodiments of the invention, the compounds presentedherein are characterized by a MIC in Gram-negative bacteria higher than200 μM and a MIC in Gram-positive bacteria higher than 20 μM.

According to another aspect of some embodiments of the presentinvention, there is provided a pharmaceutical composition which includesany one of the compounds presented herein and a pharmaceuticallyacceptable carrier.

According to some embodiments of the invention, the pharmaceuticalcomposition is packaged in a packaging material and identified in print,in or on the packaging material, for use in the treatment of a geneticdisorder.

According to another aspect of some embodiments of the presentinvention, there is provided a method for treating a genetic disorder,the method is effected by administering to a subject in need thereof atherapeutically effective amount of any one of the compounds presentedherein.

According to some embodiments of the invention, the compounds presentedherein are for use in the treatment of a genetic disorder.

According to another aspect of some embodiments of the presentinvention, there is provided a use of any one of the compounds presentedherein in the manufacture of a medicament for treating a geneticdisorder.

According to some embodiments of the invention, the genetic disorder isassociated with a premature stop codon mutation and/or a proteintruncation phenotype.

According to some embodiments of the invention, the genetic disorder isselected from the group consisting of cystic fibrosis (CF), Duchennemuscular dystrophy (DMD), ataxia-telangiectasia, Hurler syndrome,hemophilia A, hemophilia B, Usher syndrome and Tay-Sachs.

According to some embodiments of the invention, the genetic disorder iscystic fibrosis.

According to another aspect of some embodiments of the presentinvention, there is provided a process of preparing the compoundpresented herein, the process is effected by:

(a) providing a donor compound having the general Formula II:

wherein:

R₁ is selected from the group consisting of alkyl, cycloalkyl and aryl;

R₄ is hydrogen or a donor amino-protecting group;

R₅ is a donor amino-protecting group if R₄ is hydrogen or hydrogen if R₄is a donor amino-protecting group;

each of HPd is a donor hydroxyl-protecting group; and

L is a leaving group;

(b) coupling the donor compound with an acceptor compound having thegeneral formula III

wherein:

the dashed line indicates an R configuration or an S configuration;

R₃ is selected from the group consisting of hydrogen, alkyl, cycloalkyland aryl;

R6 is an acceptor amino-protecting group or(S)-4-azido-2-O-acetyl-1-butyryl;

HPa is an acceptor hydroxyl-protecting group; and

APa is an acceptor amino-protecting group; and

(c) removing each of the amino-protecting group and thehydroxyl-protecting group, thereby obtaining the compound.

According to some embodiments of the invention, the leaving group istrichloroacetimidate.

According to some embodiments of the invention, the donorhydroxyl-protecting group is O-benzoyl and the donor amino-protectinggroup is azido.

According to some embodiments of the invention, the acceptorhydroxyl-protecting group is O-acetyl and the acceptor amino-protectinggroup is azido.

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the invention, exemplarymethods and/or materials are described below. In case of conflict, thepatent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

Some embodiments of the invention are herein described, by way ofexample only, with reference to the accompanying drawings. With specificreference now to the drawings in detail, it is stressed that theparticulars shown are by way of example and for purposes of illustrativediscussion of embodiments of the invention. In this regard, thedescription taken with the drawings makes apparent to those skilled inthe art how embodiments of the invention may be practiced.

In the drawings:

FIGS. 1A-1C present a synthetic pathway for preparing C5-diasteromericesters (R,X)-27 and (S,X)-28, according to some embodiments of theinvention, wherein “a” represents DCC, 4-DMAP, CSA, DCM, at roomtemperature (FIG. 1A); ¹H NMR spectra of (R,X)-27 and (S,X)-28, whereinthe chemical shift differences (□□) between particular protons of(R,X)-27 and (S,X)-28 are highlighted (FIG. 1B); and an assignment ofabsolute configuration at the 5-carbon (denoted by X) of the majoralcohol Compound 9 by Sector rule (FIG. 1C);

FIGS. 2A-2F present the results of the stop codon readthrough assayshowing comparative graphs of in vitro stop codon suppression levelsinduced by the previously reported NB30 (marked by empty circles), byexemplary compounds according to some embodiments of the presentinvention, NB118 (marked by black triangles) and NB119 (marked by emptytriangles), and by the control drug gentamicin (marked by blackrectangles) in a series of nonsense mutation context constructsrepresenting various genetic diseases (in parenthesis), wherein resultspertaining to the R3X (USH1) construct are shown in FIG. 2A, R245X(USH1) in FIG. 2B, G542X (CF) in FIG. 2C, W1282X (CF) in FIG. 2D, Q70X(HS) in FIG. 2E, and R3381X (DMD) in FIG. 2F;

FIGS. 3A-3F present the results of the stop codon readthrough assayshowing comparative graphs of in vitro stop codon suppression levelsinduced by the previously reported NB54 (marked by black circles), byexemplary compounds according to some embodiments of the presentinvention, NB122 (marked by black triangles) and NB123 (marked by emptytriangles), and by gentamicin as a control drug (marked by blackrectangles) in a series of nonsense mutation context constructsrepresenting various genetic diseases (in parenthesis), wherein resultspertaining to the R3X (USH1) construct are shown in FIG. 3A, R245X(USH1) in FIG. 3B, G542X (CF) in FIG. 3C, W1282X (CF) in FIG. 3D, Q70X(HS) in FIG. 3E, and R3381X (DMD) in FIG. 3F;

FIGS. 4A-4D present ex vivo suppression of the PCDH15-R3X (FIG. 4A),PCDH15-R245X (FIG. 4B), IDUA-Q70X (FIG. 4C), and CFTR-W1282X (FIG. 4D)nonsense mutations, effected by the previously reported NB54 (marked byblack circles), by exemplary compounds according to some embodiments ofthe present invention, NB122 (marked by black triangle) and NB123(marked by empty triangles) and by the control drug gentamicin (markedby black rectangles);

FIGS. 5A-5D present comparative plots of the results of in vitropremature stop codon mutation suppression assays of the CFTR-G542X(FIGS. 5A and 5C), and CFTR-W 1282X (FIG. 5B and D) effected byexemplary compounds according to some embodiments of the presentinvention, NB124 (marked by black circles), NB125 (marked by emptycircles), NB127 (marked by black triangles), and NB128 (marked by emptytriangles), by the previously reported NB74 (marked by empty rhombs) andNB84 (marked by black rhombs), and by the control drugs gentamicin(marked by black rectangles) and G418 (marked by empty rectangles);

FIGS. 6A-6F present the results of the stop codon readthrough assayshowing comparative graphs of in vitro stop codon readthrough levelsinduced by exemplary compounds according to some embodiments of thepresent invention, NB124 (marked by black circles) and NB125 (marked byempty circles), by the previously reported NB74 (marked by empty rhombs)and by the control drug gentamicin (marked by black rectangles), in aseries of nonsense mutation context constructs representing variousgenetic diseases (in parenthesis), wherein results pertaining to the R3X(USH1) construct are shown in FIG. 6A, R245X (USH1) in FIG. 6B, G542X(CF) in FIG. 6C, W1282X (CF) in FIG. 6D, Q70X (HS) in FIG. 6E, andR3381X (DMD) in FIG. 6F;

FIGS. 7A-7F present the results of the stop codon readthrough assayshowing comparative graphs of in vitro stop codon suppression levelsinduced by the previously reported NB84 (marked by black rhombs), byexemplary compounds according to some embodiments of the presentinvention, NB127 (marked by black triangles) and NB128 (marked by emptytriangles), and by the control drugs G418 (marked by empty rectangles)and gentamicin (marked by black rectangles), in a series of nonsensemutation context constructs representing various genetic diseases (inparenthesis), wherein results pertaining to the R3X (USH1) construct areshown in FIG. 7A, R245X (USH1) in FIG. 7B, G542X (CF) in FIG. 7C, W1282X(CF) in FIG. 7D, Q70X (HS) in FIG. 7E, and R3381X (DMD) in FIG. 7F;

FIGS. 8A-8D present comparative plots of results of ex vivo prematurestop codon mutation suppression assays conducted for the constructsCFTR-G542X (FIGS. 8A and 8C) and CFTR-W1282X (FIGS. 8B and 8D), effectedby exemplary compounds according to some embodiments of the presentinvention, NB124 (marked by black circles), NB125 (marked by emptycircles), NB127 (marked by black triangles) and NB128 (marked by emptytriangles), by the previously reported NB74 (marked by empty rhombs) andNB84 (marked by black rhombs), and by the control drugs gentamicin(marked by black rectangles) and G418 (marked by empty rectangles);

FIGS. 9A-9E present the results of the stop codon readthrough assayshowing comparative graphs of ex vivo stop codon suppression levelsinduced by exemplary compounds according to some embodiments of thepresent invention, NB124 (marked by black circles) and NB125 (marked byempty circles), by the previously reported NB74 (marked by blackrhombs), and by the control drugs gentamicin (marked by blackrectangles) and G418 (marked by empty rectangles) in a series ofnonsense mutation context constructs representing various geneticdiseases (in parenthesis), wherein results pertaining to the R3X (USH1)construct are shown in FIG. 9A, R245X (USH1) in FIG. 9B, Q70X (HS) inFIG. 9C, W1282X (CF) in FIG. 9D and G542X (CF) in FIG. 9E;

FIGS. 10A-10E present the results of the stop codon readthrough assayshowing comparative graphs of ex vivo stop codon suppression levelsinduced by exemplary compounds according to some embodiments of thepresent invention, NB127 (marked by black rectangles) and NB128 (markedby empty triangles), by the previously reported NB84 (marked by blackrhombs) and by the control drugs gentamicin (marked by black rectangles)and G418 (marked by empty rectangles), in a series of nonsense mutationcontext constructs representing various genetic diseases (inparenthesis), wherein results pertaining to the R3X (USH1) construct areshown in FIG. 10A, R245X (USH1) in FIG. 10B, Q70X (HS) in FIG. 10C,W1282X (CF) in FIG. 10D and G542X (CF) in FIG. 10E;

FIGS. 11A-11D present semi-logarithmic plots of in vitro translationinhibition in prokaryotic (marked by black circles) and eukaryotic(marked by empty circles) systems measured for the exemplary compoundsaccording to some embodiments of the present invention, NB118 (FIG.11A), NB119 (FIG. 11B) NB122 (FIG. 11C) and NB123 (FIG. 11D);

FIGS. 12A-12D present semi-logarithmic plots of the percentages of exvivo cell viability versus concentration of the tested compound inHEK-293 (FIG. 12A and FIG. 12C) and in human foreskin fibroblasts (HFF)(FIG. 12B and FIG. 12D) cells, for gentamicin (marked by emptyrectangles), and for exemplary compounds according to some embodimentsof the present invention, NB118 (marked by empty circles), NB119 (markedby black circles), NB122 (marked by empty triangles), and NB123 (markedby black triangle); and

FIGS. 13A-13B present scatter plots for identifying possible correlationbetween readthrough activity and protein translation inhibition in vitroin eukaryotic systems as observed for a series of known compounds andexemplary compounds according to some embodiments of the presentinvention, wherein increasing inhibition of protein synthesis (lowerIC₅₀ values) is associated with the increase of readthrough activity,whereas FIG. 13A is a semilogarithmic plot of eukaryotic inhibition oftranslation versus in vitro readthrough activity at 1.4 μM concentrationof the tested aminoglycosides (shown on the X-axis) using six differentnonsense mutations (W1282X, Q70X, R3X, R245X, G542X and R3381X) and FIG.1B is a linear plot of the same data presented in FIG. 13A.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to a newclass of aminoglycosides and more particularly, but not exclusively, tonovel aminoglycosides with improved efficacy towards treatment ofgenetic disorders.

Specifically, the present invention, in some embodiments thereof,relates to a new class of compounds, derived from paromomycin, whichexhibit high premature stop codon mutations readthrough activity whileexerting low toxicity in mammalian cells. The present invention is thusfurther of pharmaceutical compositions containing these compounds, andof uses thereof in the treatment of genetic disorders, such as cysticfibrosis (CF). The present invention is further of processes ofpreparing these compounds.

The principles and operation of the present invention may be betterunderstood with reference to the figures and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details set forth in the following description or exemplified bythe Examples. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting.

As discussed above, several structural manipulations on the structure ofparomamine have given rise to synthetic aminoglycosides which have beenshown to exert improved premature stop-codon mutations readthroughactivity while exerting low toxicity in mammalian cells. Following thesestructural manipulations has lead to the development of the exemplarycompounds NB30 and NB54 as pseudo-trisaccharide derivatives of theclinical aminoglycoside paromomycin. The structural concept demonstratedin NB30 exhibited significantly reduced cytotoxicity in comparison togentamicin and paromomycin, and promoted dose-dependent suppression ofnonsense mutations of the PCDH15 gene, the underlying cause of type 1Usher syndrome (USH1), but its suppression potency was notably lowerrelative to that of gentamicin and paromomycin. NB54, which wasdeveloped as the second-generation concept structure, exhibitedsignificantly reduced cell, cochlear and acute toxicities, and hassubstantially higher readthrough efficiency than those of gentamicin andparomomycin.

While further deciphering the structure-activity relationship of suchaminoglycosides, in an attempt to further improve their therapeuticeffect in the context of genetic disorders, the present inventors haveinvestigated numerous additional modifications, at varying positions ofthe paromamine structure, and have surprisingly found that bysubstituting a hydrogen on the 5″ side-chain on the ribosamine ring(ring III) with a methyl group, the resulting aminoglycoside showsignificantly reduced cell toxicity while in parallel exhibitsubstantially higher readthrough activity of disease-causing nonsensemutations, even when compared to those of gentamicin. Hence, the presentinventors have identified another significant position in thepharmacophore that constitutes viable drug candidates that can fightdiseases that stem from genetic mutation.

Without being bound to any particular theory, it is suggested thatintroducing a modification at the ribosamine ring (Ring III) preservesalready well established impacts of the rings I and II in the previousconcept structures (see, for example, compounds NB30 and NB54 describedsupra), while introducing a new structural motif with significantsuppression activity and reduced toxicity.

While reducing the present invention to practice, the present inventorshave successfully prepared aminoglycosides (e.g., NB30, NB54 NB74 andNB84) to which the side-chain (S)-5″-methyl group was introduced to aribosamine ring (ring III), and have thereby generated a new family ofaminoglycosides. The present inventors have demonstrated that thesenewly designed compounds show significantly reduced cell toxicity whilein parallel exhibit substantially higher readthrough activity ofdisease-causing nonsense mutations, as compared, for example, togentamicin. It was also observed that the installation of (S)-5″-methylgroup does not affect cell toxicity significantly, while it greatlyenhances the stop-codon readthrough activity and specificity to theeukaryotic ribosome of the resulted structures in comparison to those ofthe previously reported structures.

Since the installation of a methyl group at C5″-position of theribosamine ring generates a new stereogenic center, the presentinventors have prepared both C5″-diastereomers with defined absoluteconfiguration and compared their biological properties.

Hence, a new pharmacophore point, (S)-5″-methyl group, has beendiscovered as a valuable structural element of the ribosamine ring (ringIII) that significantly affects suppression activity and has nosignificant influence on cell toxicity.

This new pharmacophore point is a fifth point now added to the previousfour points discovered and disclosed in, for example, WO 2007/113841.Scheme 1 presents the paromamine core with all five pharmacophore pointsdiscovered hitherto, numbered i-iv according to the sequence of theirdiscovery. Specifically, the pharmacophore point denoted “i” refers tothe provision of a hydroxyl group in position 6′; the point denoted “ii”refers to the provision of an AHB group in position N1; point “iii”refers to the provision of a third saccharide moiety (Ring III) attachedto the second saccharide ring; “iv” is the provision of a modificationat position 6′ (exemplified in Scheme 1 as a lower alkyl); and thepharmacophore point disclosed herein is denoted “v” and refers to theprovision of modification at position 5″ (exemplified in Scheme 1 as alower alkyl).

Hence, according to an aspect of embodiments of the present invention,there is provided a compound having the general formula I:

or a pharmaceutically acceptable salt thereof,

wherein:

R₁ is selected from the group consisting of alkyl, cycloalkyl and aryl,and is preferably alkyl;

R₂ is hydrogen or (S)-4-amino-2-hydroxybutyryl (AHB);

R₃ is selected from the group consisting of hydrogen, alkyl, cycloalkylor aryl, and is preferably hydrogen or alkyl; and

a stereo-configuration of each of position 6′ and position 5″ isindependently an R configuration or an S configuration.

It is noted herein that while the position of Ring III at position 05 onRing II has been shown to exhibit optimal results, other positions forRing III are contemplated, such as position O6 on Ring II and positions3′ and 4′ on Ring I.

The terms “hydroxyl” or “hydroxy”, as used herein, refer to an —OHgroup.

As used herein, the term “amine” describes a —NR′R″ group where each ofR′ and R″ is independently hydrogen, alkyl, cycloalkyl, heteroalicyclic,aryl or heteroaryl, as these terms are defined herein.

As used herein, the term “alkyl” describes an aliphatic hydrocarbonincluding straight chain and branched chain groups. The alkyl may have 1to 20 carbon atoms, or 1-10 carbon atoms, and may be branched orunbranched. According to some embodiments of the present invention, thealkyl is a low alkyl, having 1-4 carbon atoms (namely, methyl, ethyl,propyl and butyl).

The term “cycloalkyl” refers to an all-carbon monocyclic or fused ring(i.e., rings which share an adjacent pair of carbon atoms), branched orunbranched group containing 3 or more carbon atoms where one or more ofthe rings does not have a completely conjugated pi-electron system, andmay further be substituted or unsubstituted. Exemplary cycloalkyl groupsinclude, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,or cyclododecyl.

Whenever a numerical range; e.g., “1-10”, is stated herein, it impliesthat the group, in this case the alkyl group, may contain 1 carbon atom,2 carbon atoms, 3 carbon atoms, etc., up to and including 10 carbonatoms. In some embodiments, the alkyl is a lower alkyl, including 1-6 or1-4 carbon atoms. An alkyl can be substituted or unsubstituted. Whensubstituted, the substituent can be, for example, an alkyl (forming abranched alkyl), an alkenyl, an alkynyl, a cycloalkyl, an aryl, aheteroaryl, a halo, a hydroxy, an alkoxy and a hydroxyalkyl as theseterms are defined hereinbelow. The term “alkyl”, as used herein, alsoencompasses saturated or unsaturated hydrocarbon, hence this termfurther encompasses alkenyl and alkynyl.

The term “alkenyl” describes an unsaturated alkyl, as defined herein,having at least two carbon atoms and at least one carbon-carbon doublebond, e.g., allyl, vinyl, 3-butenyl, 2-butenyl, 2-hexenyl andi-propenyl. The alkenyl may be substituted or unsubstituted by one ormore substituents, as described hereinabove.

The term “alkynyl”, as defined herein, is an unsaturated alkyl having atleast two carbon atoms and at least one carbon-carbon triple bond. Thealkynyl may be substituted or unsubstituted by one or more substituents,as described hereinabove.

The term “aryl” describes an all-carbon monocyclic or fused-ringpolycyclic (i.e., rings which share adjacent pairs of carbon atoms)groups having a completely conjugated pi-electron system. The aryl groupmay be substituted or unsubstituted by one or more substituents, asdescribed hereinabove.

The term “heteroaryl” describes a monocyclic or fused ring (i.e., ringswhich share an adjacent pair of atoms) group having in the ring(s) oneor more atoms, such as, for example, nitrogen, oxygen and sulfur and, inaddition, having a completely conjugated pi-electron system. Examples,without limitation, of heteroaryl groups include pyrrole, furane,thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine,quinoline, isoquinoline and purine. The heteroaryl group may besubstituted or unsubstituted by one or more substituents, as describedhereinabove. Representative examples are thiadiazole, pyridine, pyrrole,oxazole, indole, purine and the like.

The term “heteroalicyclic”, as used herein, describes a monocyclic orfused ring group having in the ring(s) one or more atoms such asnitrogen, oxygen and sulfur. The rings may also have one or more doublebonds. However, the rings do not have a completely conjugatedpi-electron system. The heteroalicyclic may be substituted orunsubstituted. Substituted heteroalicyclic may have one or moresubstituents, whereby each substituent group can independently be, forexample, alkyl cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl andheteroalicyclic. Representative examples are morpholine, piperidine,piperazine, tetrahydrofurane, tetrahydropyrane and the like.

The term “halide”, as used herein, refers to the anion of a halo atom,i.e. F⁻, Cl⁻, Br⁻ and I⁻.

The term “halo” refers to F, Cl, Br and I atoms as substituents.

The term “alkoxy” refers to an R′—O⁻ anion, wherein R′ is as definedhereinabove.

The term “hydroxyalkyl,” as used herein, refers to an alkyl groupsubstituted with one hydroxy group, e.g., hydroxymethyl, p-phydroxyethyland 4-hydroxypentyl.

The term “alkoxyalkyl,” as used herein, refers to an alkyl groupsubstituted with one alkoxy group, e.g., methoxymethyl, 2-methoxyethyl,4-ethoxybutyl, n-propoxyethyl and t-butylethyl.

The moiety (S)-4-amino-2-hydroxybutyryl, is also referred to herein asAHB. According to some embodiments of the present invention, analternative to the AHB moiety can be the α-hydroxy-β-aminopropionyl(AHP) moiety. These so-called side chains or optional moieties arebelieved to block the access of aminoglycoside-modifying enzymes to thetarget sites. Moreover, AHB or AHP contain a 1,3- or 1,2-hydroxylaminemoiety that binds to phosphodiesters and to the hoogsten base face ofguanosine of the A-site of 16S rRNA. It is noted herein that accordingto some embodiments of the present invention, other moieties whichinvolve a combination of carbonyl(s), hydroxyl(s) and amino group(s)along a lower alkyl exhibiting any stereochemistry, are contemplated asoptional substituents in place of AHB and/or AHP. For example,2-amino-3-hydroxybutanoyl, 3-amino-2-hydroxypentanoyl,5-amino-3-hydroxyhexanoyl and the likes.

Herein, it is to be understood that whenever reference is made to AHB,equivalent groups as described herein (e.g., AHP) are also encompassed.

As used herein, the phrase “moiety” describes a part, and preferably amajor part, of a chemical entity, such as a molecule or a group, whichhas underwent a chemical reaction and is now covalently linked toanother molecular entity.

According to some embodiments of the present invention, R₁ is alkyl.

According to some embodiments, R₁ is a lower alkyl as defined herein,including, but not limited to, methyl, ethyl, propyl, butyl, andisopropyl. According to other embodiments of the present invention, R₁is methyl.

Alternatively, R₁ is cycloalkyl, including, but not limited to,cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

Further alternatively, R₁ is aryl, such as substituted or unsubstitutedphenyl. Non-limiting examples include phenyl and toluene.

In some embodiments of the present invention, R₁ is alkyl, as describedherein, and R₂ and R₃ are each hydrogen. In terms of the pharmacophorepoints presented in Scheme 1 (vide supra), these compounds possess thefifth (v) point and do not possess the second (ii) and fourth (iv)points. These compounds exhibit superior pharmacologic profile comparedto previously known compounds and drugs which are considered for use intreating genetic disorders, namely these compounds are less toxic andmore efficient in reading-through premature stop codon mutations, asdemonstrated in the Examples section that follows below.

Exemplary aminoglycoside compounds which exhibit hydrogen in positionsR₂ and R₃ include:

which differ from each other in the stereo-configuration of the chiralcenter at position 5″ of Ring III.

Optionally, R₁ is cycloalkyl, as described herein, and R₂ and R₃ areeach hydrogen.

Optionally, R₁ is aryl, as described herein, and R₂ and R₃ are eachhydrogen.

In some embodiments of the present invention, R₁ is alkyl, as describedherein, R₂ is AHB and R₃ is a hydrogen atom. In terms of thepharmacophore points presented in Scheme 1 (vide supra), other thanpossessing the fifth (v) point, these compounds possess the second (ii)point and do not possess the fourth (iv) point. These compounds exhibitsuperior pharmacologic profile compared to previously known compoundsand drugs which are considered for use in treating genetic disorders,namely these compounds are less toxic and more efficient inreading-through premature stop codon mutations, as demonstrated in theExamples section that follows below.

Exemplary aminoglycoside compounds having an AHB moiety at position R₂and hydrogen in R₃ include:

which differ from each other in the stereo-configuration of the chiralcenter at position 5″ of Ring III.

Optionally, R₁ is cycloalkyl, as described herein, R₂ is AHB and R₃ is ahydrogen atom.

Optionally, R₁ is aryl, as described herein, R₂ is AHB and R₃ is ahydrogen atom.

In some embodiments of the present invention, R₁ is alkyl, as describedherein, R₂ is hydrogen and R₃ is alkyl. In terms of the pharmacophorepoints presented in Scheme 1 (vide supra), other than possessing thefifth (v) point, these compounds do not possess the second (ii) pointand do possess the fourth (iv) point. These compounds exhibit superiorpharmacologic profile compared to previously known compounds and drugswhich are considered for use in treating genetic disorders, namely thesecompounds are less toxic and more efficient in reading-through prematurestop codon mutations, as demonstrated in the Examples section thatfollows below.

Optionally, R₁ is cycloalkyl, as described herein, R₂ is hydrogen and R₃is alkyl.

Optionally, R₁ is aryl, as described herein, R₂ is hydrogen and R₃ isalkyl.

According to some embodiments of the present invention, in any of theabove-described embodiments where R₃ is alkyl, R₃ is a lower alkyl, asdefined herein. According to these embodiments, R₃ is methyl.

Optionally, R₁ is alkyl, as described herein, R₂ is hydrogen and R₃ iscycloalkyl.

Optionally, R₁ is cycloalkyl, as described herein, R₂ is hydrogen and R₃is cycloalkyl.

Optionally, R₁ is aryl, as described herein, R₂ is hydrogen and R₃ iscycloalkyl.

Optionally, R₁ is alkyl, as described herein, R₂ is hydrogen and R₃ isaryl.

Optionally, R₁ is cycloalkyl, as described herein, R₂ is hydrogen and R₃is aryl.

Optionally, R₁ is aryl, as described herein, R₂ is hydrogen and R₃ isaryl.

Exemplary aminoglycoside compounds which exhibit hydrogen in position R₂and alkyl in position R₃ include:

which differ from each other in the stereo-configuration of the chiralcenter at position 5″ of Ring III.

In some embodiments of the present invention, R₂ is AHB and R₃ is alkyl.In terms of the pharmacophore points presented in Scheme 1 (vide supra),these compounds possess all five points; These compounds exhibit themost superior pharmacologic profile compared to previously knowncompounds and drugs in terms of lower cytotoxicity and higher inreadthrough efficiency, as demonstrated in the Examples section thatfollows below.

Exemplary aminoglycoside compounds wherein R₂ is AHB and R₃ is alkylinclude:

which differ from each other in the stereo-configuration of the chiralcenter at position 5″ of Ring III.

Optionally, R₁ is cycloalkyl, as described herein, R₂ is AHB and R₃ isalkyl. Optionally, R₁ is aryl, as described herein, R₂ is AHB and R₃ isalkyl.

According to some embodiments of the present invention, in any of theabove-described embodiments where R₃ is alkyl, R₃ is a lower alkyl, asdefined herein. According to these embodiments, R₃ is methyl.

Optionally, R₁ is alkyl, as described herein, R₂ is AHB and R₃ iscycloalkyl.

Optionally, R₁ is cycloalkyl, as described herein, R₂ is AHB and R₃ iscycloalkyl.

Optionally, R₁ is aryl, as described herein, R₂ is AHB and R₃ iscycloalkyl.

Optionally, R₁ is alkyl, as described herein, R₂ is AHB and R₃ is aryl.

Optionally, R₁ is cycloalkyl, as described herein, R₂ is AHB and R₃ isaryl.

Optionally, R₁ is aryl, as described herein, R₂ is AHB and R₃ is aryl.

While searching for a way to predict and evaluate quantitatively thecapacity of a synthetic aminoglycoside to constitute a drug candidatefor treating genetic diseases caused by premature stop codon mutations(exhibit readthrough activity) and at the same time exhibit low or nocytotoxicity, it was found that high selectivity of the compound toeukaryotic cytoplasmic translation systems (i.e., eukaryotic cytoplasmicribosomes) compared to prokaryotic translation systems, which aresimilar or resembles to some extent the mitochondrial translationsystem, can be used as a predictive measure. A numeric value that canreadily be used to quantify this selectivity is the ratio IC₅₀^(Euk)/IC₅₀ ^(Pro) which correlates an inhibition of translation ineukaryotes to inhibition of translation in prokaryotes (see, Table 3hereinbelow). As demonstrated in the Examples section below, a notableselectivity of any given aminoglycoside compound, such as the compoundsaccording to some embodiments of the present invention, towardsinhibiting translation in eukaryote over inhibiting translation inprokaryote can be used to predict its effectiveness and safety as a drugcandidate for treating genetic disorders associated with premature stopcodon mutations.

Nonetheless, it is noted herein that the IC₅₀ ^(Euk)/IC₅₀ ^(Pro) ratiowhich indicates selectivity, is not a sufficient criteria for selectingdrug candidates from this family of aminoglycosides; one must alsoconsider the mechanism of translation inhibition. For example, it wasfound that the aminoglycoside NB33, which is a dimer of the parentcompound paromamine, exhibits a ratio value of about 2, which isregarded as low and thus predictive for a good readthrough drugcandidate. However, NB33 exhibits essentially no readthrough activity.It is assumed that NB33 inhibits the translation mechanism in adifferent inhibition mode, as shown in the crystal of complex betweenthe cytoplasmic A site RNA and NB33 [Chem. Bio. Chem., 2007, 8(14), p.1617].

Without being bound by any particular theory, one possible conclusionfrom the above discussion is that for an aminoglycoside to exhibitdesired traits of a premature stop codon mutation readthrough drugcandidate, 1) it should inhibit both prokaryotic and eukaryoticribosomes by same mechanism of to binding to the aminoacyl-tRNA bindingsite and stabilizing the decoding conformation, or inhibit proteintranslation process by interfering with the fidelity of proof-readingprocess; and 2) the IC₅₀ ^(Euk)/IC₅₀ ^(Pro) ratio favoring eukaryotesshould also be accompanied with a significant decrease in thespecificity of the compound to the prokaryotic ribosome; in other wordselevated IC₅₀ ^(Pro) values. A representative example for thisrequirement is G418; it IC₅₀ ^(Euk)/IC₅₀ ^(Pro) ratio is 225, which issignificantly lower to that of gentamicin but still it is highly toxicas indicated by a relatively very low IC₅₀ ^(Pro) value.

Thus, according to some embodiments of the present invention, thecompounds presented herein are characterized by a ratio of IC₅₀translation inhibition in eukaryotes to IC₅₀ translation inhibition inprokaryotes lower than 15, lower than 10, lower than 5 or lower than 1,including any intermediate value between 15 and 1.

As demonstrated hereinbelow, while preparing and testing exemplarycompounds according to some embodiments of the present invention, it hasbeen observed that the increased inhibition of prokaryotic cytoplasmicprotein synthesis is also associated with increased readthroughactivity. Data presented in Table 3 shows that the systematic additionof points of the pharmacophore presented in Scheme 1 gradually increasesthe specificity of compounds to the cytoplasmic ribosome and decreasetheir specificity to the prokaryotic ribosome.

It would be reasonable to expect aminoglycosides to be selective towardsprokaryotes, since aminoglycosides have developed by natural selectionin Streptomyces genus and other species such as speciesSaccharopolyspora erythraea, to be active against other prokaryotes.Nonetheless, compounds according to some embodiments of the presentinvention, exhibit reversed selectivity to eukaryotic versus prokaryotictranslation systems (ribosome).

Thus, according to some embodiments of the present invention, thecompounds presented herein are characterized by a ratio of IC₅₀translation inhibition in eukaryotes to IC₅₀ translation inhibition inprokaryotes lower than 15, lower than 1.

As discussed hereinabove, a promising aminoglycoside compound, accordingto some embodiments of the present invention, is one that does not havea notable or any antimicrobial activity. Such non-activity is alsopredictive for low or no cytotoxicity of the compound to mammalians. Theresults, which show that the exemplary compounds which have beenprepared and tested for antimicrobial activity or lack thereof, arepresented in Tables 1 and 2 hereinbelow.

Hence, according to some embodiments of the present invention, thecompounds presented herein are characterized by a MIC value inGram-negative bacteria which is higher than 200 μM, higher than 300 μM,higher than 500 μM, higher than 700 μM, or higher than 1000 μM, as wellas a MIC value in Gram-positive bacteria which is higher than 20 μM,higher than 40 μM, higher than 80 μM, or higher than 100 μM.

The present embodiments further encompass any enantiomers,diastereomers, prodrugs, solvates, hydrates and/or pharmaceuticallyacceptable salts of the compounds described herein.

As used herein, the term “enantiomer” refers to a stereoisomer of acompound that is superposable with respect to its counterpart only by acomplete inversion/reflection (mirror image) of each other. Enantiomersare said to have “handedness” since they refer to each other like theright and left hand. Enantiomers have identical chemical and physicalproperties except when present in an environment which by itself hashandedness, such as all living systems. In the context of the presentembodiments, a compound may exhibit one or more chiral centers, each ofwhich exhibiting an R— or an S-configuration and any combination, andcompounds according to some embodiments of the present invention, canhave any their chiral centers exhibit an R— or an S-configuration.

The term “diastereomers”, as used herein, refers to stereoisomers thatare not enantiomers to one another. Diastereomerism occurs when two ormore stereoisomers of a compound have different configurations at one ormore, but not all of the equivalent (related) stereocenters and are notmirror images of each other. When two diastereoisomers differ from eachother at only one stereocenter they are epimers. Each stereo-center(chiral center) gives rise to two different configurations and thus totwo different stereoisomers. In the context of the present invention,embodiments of the present invention encompass compounds with multiplechiral centers that occur in any combination of stereo-configuration,namely any diastereomer.

The term “prodrug” refers to an agent, which is converted into theactive compound (the active parent drug) in vivo. Prodrugs are typicallyuseful for facilitating the administration of the parent drug. They may,for instance, be bioavailable by oral administration whereas the parentdrug is not. A prodrug may also have improved solubility as comparedwith the parent drug in pharmaceutical compositions. Prodrugs are alsooften used to achieve a sustained release of the active compound invivo. An example, without limitation, of a prodrug would be a compoundof the present invention, having one or more carboxylic acid moieties,which is administered as an ester (the “prodrug”). Such a prodrug ishydrolyzed in vivo, to thereby provide the free compound (the parentdrug). The selected ester may affect both the solubility characteristicsand the hydrolysis rate of the prodrug.

The term “solvate” refers to a complex of variable stoichiometry (e.g.,di-, tri-, tetra-, penta-, hexa-, and so on), which is formed by asolute (the compound of the present invention) and a solvent, wherebythe solvent does not interfere with the biological activity of thesolute. Suitable solvents include, for example, ethanol, acetic acid andthe like.

The term “hydrate” refers to a solvate, as defined hereinabove, wherethe solvent is water.

The phrase “pharmaceutically acceptable salt” refers to a chargedspecies of the parent compound and its counter ion, which is typicallyused to modify the solubility characteristics of the parent compoundand/or to reduce any significant irritation to an organism by the parentcompound, while not abrogating the biological activity and properties ofthe administered compound. An example, without limitation, of apharmaceutically acceptable salt would be a hydroxyl anion (O⁻) and acation such as, but not limited to, ammonium, sodium, potassium and thelike. Another example, without limitation, of a pharmaceuticallyacceptable salt would be an ammonium cation and an acid addition saltthereof. Examples of acid addition salts include, but are not limitedto, hydrochloric acid addition salt, sulfuric acid addition salt(sulfate salt), acetic acid addition salt, ascorbic acid addition salt,benzenesulfonic acid addition salt, camphorsulfonic acid addition salt,citric acid addition salt, maleic acid addition salt, methanesulfonicacid addition salt, naphthalenesulfonic acid addition salt, oxalic acidaddition salt, phosphoric acid addition salt, succinic acid additionsalt, sulfuric acid addition salt, tartaric acid addition salt, andtoluenesulfonic acid addition salt.

According to some embodiments of the present invention, the acidaddition salt is a sulfate salt.

Further according to the present invention, there are provided processesof preparing the compounds described herein.

The synthetic pathways described herein include a reaction between anacceptor and a donor, whereby the term “acceptor” is used herein todescribe the skeletal structure derived from paromamine which has atleast one and preferably selectively selected available (unprotected)hydroxyl group at positions such as C5, C6 and C3′, which is reactiveduring a glycosylation reaction, and can accept a glycosyl, and the term“donor” is used herein to describe the glycosyl. According to someembodiments of the present invention, the position on the acceptor isthe C5 position.

The term “glycosyl”, as used herein, refers to a chemical group which isobtained by removing the hydroxyl group from the hemiacetal function ofa monosaccharide and, by extension, of a lower oligosaccharide.

The term “monosaccharide”, as used herein and is well known in the art,refers to a simple form of a sugar that consists of a single saccharidemolecule which cannot be further decomposed by hydrolysis. Most commonexamples of monosaccharides include glucose (dextrose), fructose,galactose, and ribose. Monosaccharides can be classified according tothe number of carbon atoms of the carbohydrate, i.e., triose, having 3carbon atoms such as glyceraldehyde and dihydroxyacetone; tetrose,having 4 carbon atoms such as erythrose, threose and erythrulose;pentose, having 5 carbon atoms such as arabinose, lyxose, ribose,xylose, ribulose and xylulose; hexose, having 6 carbon atoms such asallose, altrose, galactose, glucose, gulose, idose, mannose, talose,fructose, psicose, sorbose and tagatose; heptose, having 7 carbon atomssuch as mannoheptulose, sedoheptulose; octose, having 8 carbon atomssuch as 2-keto-3-deoxy-manno-octonate; nonose, having 9 carbon atomssuch as sialose; and decose, having 10 carbon atoms. Monosaccharides arethe building blocks of oligosaccharides like sucrose (common sugar) andother polysaccharides (such as cellulose and starch).

The term “oligosaccharide” as used herein refers to a compound thatcomprises two or more monosaccharide units, as these are defined herein.According to some embodiments of the present invention, anoligosaccharide comprises 2-6 monosaccharides. Alternatively, anoligosaccharide comprises 2-4 monosaccharides, or further alternatively,an oligosaccharide is a disaccharide moiety, having two monosaccharideunits.

The donors and acceptors are designed so as to form the desiredcompounds according to some embodiments of the present invention. Thefollowing describes some embodiments of this aspect of the presentinvention, presenting exemplary processes of preparing exemplary subsetsof the compounds described herein. Detailed processes of preparingexemplary compounds according to some embodiments of the presentinvention, are presented in the Examples section that follows below.

The syntheses of the compounds according to some embodiments of thepresent invention, generally include (i) preparing an acceptor compoundby selective protection of one or more hydroxyls and amines at selectedpositions present on the paromamine scaffold, leaving one or twopositions unprotected and therefore free to accept a donor (glycosyl)compound as defined herein; (ii) preparing a donor compound by selectiveprotection of one or more hydroxyls and amines at selected positionspresent on the glycosyl, leaving one position unprotected and thereforefree to couple with an acceptor compound as defined herein; (iii)subjecting the donor and the acceptor to a coupling reaction; and (iii)removing of all protecting groups to thereby obtain the desiredcompound.

The phrase “protecting group”, as used herein, refers to a substituentthat is commonly employed to block or protect a particular functionalitywhile reacting other functional groups on the compound. For example, an“amino-protecting group” is a substituent attached to an amino groupthat blocks or protects the amino functionality in the compound.Suitable amino-protecting groups include azide (azido), N-phthalimido,N-acetyl, N-trifluoroacetyl, N-t-butoxycarbonyl (BOC),N-benzyloxycarbonyl (CBz) and N-9-fluorenylmethylenoxycarbonyl (Fmoc).Similarly, a “hydroxyl-protecting group” refers to a substituent of ahydroxyl group that blocks or protects the hydroxyl functionality.Suitable protecting groups include isopropylidene ketal andcyclohexanone dimethyl ketal (forming a 1,3-dioxane with two adjacenthydroxyl groups), 4-methoxy-1-methylbenzene (forming a 1,3-dioxane withtwo adjacent hydroxyl groups), O-acetyl, O-chloroacetyl, O-benzoyl andO-silyl. For a general description of protecting groups and their use,see T. W. Greene, Protective Groups in Organic Synthesis, John Wiley &Sons, New York, 1991.

According to some embodiments, the amino-protecting groups include anazido (N₃—) and/or an N-phthalimido group, and the hydroxyl-protectinggroups include O-acetyl (AcO-), O-benzoyl (BzO-) and/or O-chloroacetyl.It is noted herein that when applicable, a “protecting group” refers toa moiety that can protect one reactive function on a compound or morethan one function at the same time, such as in the case of two adjacentfunctionalities, e.g., two hydroxyl groups that can be protected at onceby a isopropylidene ketal.

Hence, there is provided a process of preparing the compounds having thegeneral Formula I as presented herein. The process is effected bypreparing a suitably protected acceptor compound and a suitablyprotected donor compound, coupling these two compounds to one another,and subsequently removing all the protecting groups from the resultingcompound.

The donor compound is a protected monosaccharide which can berepresented by the general Formula II, having a leaving group atposition 1″ thereof, denoted L, and an alkyl, cycloalkyl or aryl atposition 5″, denoted

wherein:

R₁ is selected from the group consisting of alkyl, cycloalkyl and aryl;

R₄ is hydrogen or a donor amino-protecting group;

R₅ is a donor amino-protecting group if R₄ is hydrogen or hydrogen if R₄is a donor amino-protecting group; and

each of HPd is a donor hydroxyl-protecting group.

It is noted herein that the absolute stereo-configuration of the chiralcenter at position 5″ is determined by the identity of R₄ and R5, givingboth options of R- and S-configuration as two individual and separabledonors (being diastereomers) or as a racemic mixture thereof. A detailedprocess for obtaining each of the R- and S-donor compounds and a methodfor assigning the absolute stereo-configuration thereof is presented inthe Examples section below.

As used herein, the phrase “leaving group” describes a labile atom,group or chemical moiety that readily undergoes detachment from anorganic molecule during a chemical reaction, while the detachment isfacilitated by the relative stability of the leaving atom, group ormoiety thereupon. Typically, any group that is the conjugate base of astrong acid can act as a leaving group. Representative examples ofsuitable leaving groups according to the present embodiments thereforeinclude, without limitation, trichloroacetimidate, acetate, tosylate,triflate, sulfonate, azide, halide, hydroxy, thiohydroxy, alkoxy,cyanate, thiocyanate, nitro and cyano.

According to some embodiments of the present invention, the leavinggroup is trichloroacetimidate, which gave the most satisfactory resultsin the coupling reaction with the acceptor, although other leavinggroups are contemplated.

According to some embodiments of the present invention, each of thedonor hydroxyl-protecting groups is O-benzoyl and the donoramino-protecting group in either R₄ or R₅ is azido, although otherprotecting groups are contemplated.

The structure of the donor compound sets the absolute structure of RingIII in the resulting compound according to some embodiments of thepresent invention, namely the stereo-configuration of the 5″ positionand the type of alkyl at that position. Exemplary donor compounds,suitable for affording compounds according to some embodiments of thepresent invention, include Compound (S)-17 and Compound (R)-18, thepreparation thereof is illustrated in Scheme 2 hereinbelow.

The acceptor, according to some embodiments, has the general FormulaIII:

wherein:

the dashed line indicates an R configuration or an S configuration;

R₃ is selected from the group consisting of hydrogen, alkyl, cycloalkyland aryl;

R6 is an acceptor amino-protecting group or(S)-4-azido-2-O-acetyl-1-butyryl (a protected form of AHB);

HPa is an acceptor hydroxyl-protecting group; and

APa is an acceptor amino-protecting group.

According to some embodiments of the present invention, the acceptorhydroxyl-protecting group is O-acetyl, and the donor amino-protectinggroup is azido, although other protecting groups are contemplated.

It is noted herein that the exemplary embodiment provided hereinaboverefers to a protected for of AHB, however it is not meant to be limitingto use of the AHB moiety as other useful moieties, such as AHP aspresented hereinabove, may be used instead. In those cases the processwill be modified by using an acceptor compound wherein the reactivegroups of the moiety used in place of AHB are protected accordingly.

The structure of the acceptor compound sets the absolute structure ofRing I and Ring II in the resulting compound according to someembodiments of the present invention, namely the stereo-configuration ofthe 6′ position and the type of alkyl at that position when present, andthe substituent on the amino group at position N1. Exemplary acceptorcompounds, suitable for affording compounds according to someembodiments of the present invention, include Compounds 19, 20, 219 and220, the preparation of which is illustrated in Scheme 3 and Scheme 4hereinbelow.

The process is therefore effected by:

(a) providing both the desired donor compound and desired acceptorcompound;

(a) coupling the aforementioned acceptor compound to the aforementioneddonor compound (also referred to as a glycosylation reaction); and

(b) subsequently removing each of the protecting groups to therebyobtain the desired compound.

For example, the exemplary compound NB118 can be afforded bydeprotecting Compound (S)-21, which is obtained by glycosilating(coupling) acceptor Compound 19 with donor Compound (S)-17.Correspondingly, the exemplary compound NB119 is obtained bydeprotecting Compound (R)-22 which is the product of coupling acceptorCompound 19 with donor Compound (R)-18.

Similarly, the exemplary compound NB122 is afforded by deprotectingCompound (S)-23, the coupling product between acceptor Compound 20 anddonor Compound (S)-17. Correspondingly, the exemplary compound NB123 isobtained by deprotecting Compound (R)-24 which is the product ofcoupling acceptor Compound 20 with donor Compound (R)-18.

The exemplary compound NB124 is afforded by deprotecting Compound(S)-221, the coupling product between acceptor Compound 219 and donorCompound (S)-17. Correspondingly, the exemplary compound NB125 isobtained by deprotecting Compound (R)-222 which is the product ofcoupling acceptor Compound 219 with donor Compound (R)-18.

The exemplary compound NB127 is afforded by deprotecting Compound(S)-223, the coupling product between acceptor Compound 220 and donorCompound (S)-17. Correspondingly, the exemplary compound NB128 isobtained by deprotecting Compound (R)-224 which is the product ofcoupling acceptor Compound 220 with donor Compound (R)-18.

As demonstrated in the Examples section that follows the compoundspresented herein were designed so as to, and were indeed shown to,possess a truncation mutation suppression activity, namely the abilityto induce readthrough of a premature stop codon mutation. Such anactivity renders these compounds suitable for use as therapeuticallyactive agents for the treatment of genetic disorders, and particularlysuch disorders which are characterized by a truncation mutation.

Thus, according to another aspect of the present invention there isprovided a method of treating a genetic disorder. The method, accordingto this aspect of the present invention, is effected by administering toa subject in need thereof a therapeutically effective amount of one ormore of the compounds presented herein having a general Formula I.

As used herein, the term “treating” includes abrogating, substantiallyinhibiting, slowing or reversing the progression of a condition,substantially ameliorating clinical or aesthetical symptoms of acondition or substantially preventing the appearance of clinical oraesthetical symptoms of a condition.

As used herein, the phrase “therapeutically effective amount” describesan amount of the polymer being administered which will relieve to someextent one or more of the symptoms of the condition being treated.

The phrase “genetic disorder”, as used herein, refers to a chronicdisorder which is caused by one or more defective genes that are ofteninherited from the parents, and which can occur unexpectedly when twohealthy carriers of a defective recessive gene reproduce, or when thedefective gene is dominant. Genetic disorders can occur in differentinheritance patterns which include the autosomal dominant patternwherein only one mutated copy of the gene is needed for an offspring tobe affected, and the autosomal recessive pattern wherein two copies ofthe gene must be mutated for an offspring to be affected.

According to some embodiments the genetic disorder involves a genehaving a truncation mutation which leads to improper translationthereof. The improper translation causes a reduction or abolishment ofsynthesis of an essential protein.

Exemplary such genetic disorders include, but are not limited to, cysticfibrosis (CF), Duchenne muscular dystrophy (DMD), ataxia-telangiectasia,Hurler syndrome, hemophilia A, hemophilia B, Usher syndrome andTay-Sachs.

Accordingly, there is provided a use of a compound having the generalFormula I as presented herein in the manufacture of a medicament fortreating a genetic disorder.

In any of the methods and uses described herein, the compounds describedherein can be utilized either per se or form a part of a pharmaceuticalcomposition, which further comprises a pharmaceutically acceptablecarrier.

Thus, further according to the present invention, there is provided apharmaceutical composition which comprises, as an active ingredient, anyof the novel compounds described herein and a pharmaceuticallyacceptable carrier.

As used herein a “pharmaceutical composition” refers to a preparation ofthe compounds presented herein, with other chemical components such aspharmaceutically acceptable and suitable carriers and excipients. Thepurpose of a pharmaceutical composition is to facilitate administrationof a compound to an organism.

Hereinafter, the term “pharmaceutically acceptable carrier” refers to acarrier or a diluent that does not cause significant irritation to anorganism and does not abrogate the biological activity and properties ofthe administered compound. Examples, without limitations, of carriersare: propylene glycol, saline, emulsions and mixtures of organicsolvents with water, as well as solid (e.g., powdered) and gaseouscarriers.

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of acompound. Examples, without limitation, of excipients include calciumcarbonate, calcium phosphate, various sugars and types of starch,cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

Techniques for formulation and administration of drugs may be found in“Remington's Pharmaceutical Sciences” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore pharmaceutically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the compounds presentedherein into preparations which, can be used pharmaceutically. Properformulation is dependent upon the route of administration chosen.

According to some embodiments, the administration is effected orally.For oral administration, the compounds presented herein can beformulated readily by combining the compounds with pharmaceuticallyacceptable carriers well known in the art. Such carriers enable thecompounds presented herein to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for oral ingestion by a patient. Pharmacological preparations for oraluse can be made using a solid excipient, optionally grinding theresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/orphysiologically acceptable polymers such as polyvinylpyrrolidone (PVP).If desired, disintegrating agents may be added, such as cross-linkedpolyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such assodium alginate.

Pharmaceutical compositions, which can be used orally, include push-fitcapsules made of gelatin as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules may contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, lubricants such as talc ormagnesium stearate and, optionally, stabilizers. In soft capsules, thecompounds presented herein may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. All formulations fororal administration should be in dosages suitable for the chosen routeof administration.

For injection, the compounds presented herein may be formulated inaqueous solutions, preferably in physiologically compatible buffers suchas Hank's solution, Ringer's solution, or physiological saline bufferwith or without organic solvents such as propylene glycol, polyethyleneglycol.

For transmucosal administration, penetrants are used in the formulation.Such penetrants are generally known in the art.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, titanium dioxide, lacquer solutions and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active am inoglicoside compounds doses.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds presented herein areconveniently delivered in the form of an aerosol spray presentation(which typically includes powdered, liquefied and/or gaseous carriers)from a pressurized pack or a nebulizer, with the use of a suitablepropellant, e.g., dichlorodifluoromethane, trichlorofluoromethane,dichloro-tetrafluoroethane or carbon dioxide. In the case of apressurized aerosol, the dosage unit may be determined by providing avalve to deliver a metered amount. Capsules and cartridges of, e.g.,gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compounds presented herein and a suitablepowder base such as, but not limited to, lactose or starch.

The compounds presented herein may be formulated for parenteraladministration, e.g., by bolus injection or continuous infusion.Formulations for injection may be presented in unit dosage form, e.g.,in ampoules or in multidose containers with optionally, an addedpreservative. The compositions may be suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the compounds preparation in water-soluble form.Additionally, suspensions of the compounds presented herein may beprepared as appropriate oily injection suspensions and emulsions (e.g.,water-in-oil, oil-in-water or water-in-oil in oil emulsions). Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acids esters such as ethyl oleate, triglycerides orliposomes. Aqueous injection suspensions may contain substances, whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension may alsocontain suitable stabilizers or agents, which increase the solubility ofthe compounds presented herein to allow for the preparation of highlyconcentrated solutions.

Alternatively, the compounds presented herein may be in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free water,before use.

The compounds presented herein may also be formulated in rectalcompositions such as suppositories or retention enemas, using, e.g.,conventional suppository bases such as cocoa butter or other glycerides.

The pharmaceutical compositions herein described may also comprisesuitable solid of gel phase carriers or excipients. Examples of suchcarriers or excipients include, but are not limited to, calciumcarbonate, calcium phosphate, various sugars, starches, cellulosederivatives, gelatin and polymers such as polyethylene glycols.

Pharmaceutical compositions suitable for use in context of the presentinvention include compositions wherein the active ingredients arecontained in an amount effective to achieve the intended purpose. Morespecifically, a therapeutically effective amount means an amount ofcompounds presented herein effective to prevent, alleviate or amelioratesymptoms of the disorder, or prolong the survival of the subject beingtreated.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For any compounds presented herein used in the methods of the presentembodiments, the therapeutically effective amount or dose can beestimated initially from activity assays in animals. For example, a dosecan be formulated in animal models to achieve a circulatingconcentration range that includes the mutation suppression levels asdetermined by activity assays (e.g., the concentration of the testcompounds which achieves a substantial read-through of the truncationmutation). Such information can be used to more accurately determineuseful doses in humans.

Toxicity and therapeutic efficacy of the compounds presented herein canbe determined by standard pharmaceutical procedures in experimentalanimals, e.g., by determining the EC₅₀ (the concentration of a compoundwhere 50% of its maximal effect is observed) and the LD₅₀ (lethal dosecausing death in 50% of the tested animals) for a subject compound. Thedata obtained from these activity assays and animal studies can be usedin formulating a range of dosage for use in human.

The dosage may vary depending upon the dosage form employed and theroute of administration utilized. The exact formulation, route ofadministration and dosage can be chosen by the individual physician inview of the patient's condition. (See e.g., Fingl et al., 1975, in “ThePharmacological Basis of Therapeutics”, Ch. 1 p.1).

Dosage amount and interval may be adjusted individually to provideplasma levels of the compounds presented herein which are sufficient tomaintain the desired effects, termed the minimal effective concentration(MEC). The MEC will vary for each preparation, but can be estimated fromin vitro data; e.g., the concentration of the compounds necessary toachieve 50-90% expression of the whole gene having a truncationmutation, i.e. read-through of the mutation codon. Dosages necessary toachieve the MEC will depend on individual characteristics and route ofadministration. HPLC assays or bioassays can be used to determine plasmaconcentrations.

Dosage intervals can also be determined using the MEC value.Preparations should be administered using a regimen, which maintainsplasma levels above the MEC for 10-90% of the time, preferable between30-90% and most preferably 50-90%.

Depending on the severity and responsiveness of the chronic condition tobe treated, dosing can also be a single periodic administration of aslow release composition described hereinabove, with course of periodictreatment lasting from several days to several weeks or until sufficientamelioration is effected during the periodic treatment or substantialdiminution of the disorder state is achieved for the periodic treatment.

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc. Compositions of the present invention may, if desired, be presentedin a pack or dispenser device, such as an FDA (the U.S. Food and DrugAdministration) approved kit, which may contain one or more unit dosageforms containing the active ingredient. The pack may, for example,comprise metal or plastic foil, such as, but not limited to a blisterpack or a pressurized container (for inhalation). The pack or dispenserdevice may be accompanied by instructions for administration. The packor dispenser may also be accompanied by a notice associated with thecontainer in a form prescribed by a governmental agency regulating themanufacture, use or sale of pharmaceuticals, which notice is reflectiveof approval by the agency of the form of the compositions for human orveterinary administration. Such notice, for example, may be of labelingapproved by the U.S. Food and Drug Administration for prescription drugsor of an approved product insert. Compositions comprising a compoundaccording to the present embodiments, formulated in a compatiblepharmaceutical carrier may also be prepared, placed in an appropriatecontainer, and labeled for treatment of an indicated condition ordiagnosis, as is detailed hereinabove.

Thus, in some embodiments, the pharmaceutical composition is packaged ina packaging material and identified in print, in or on the packagingmaterial, for use in the treatment of a genetic disorder, as definedherein.

In any of the composition, methods and uses described herein, thecompounds can be utilized in combination with other agents useful in thetreatment of the genetic disorder.

Being primarily directed at treating genetic disorders, which arechronic by definition, the compounds presented herein or pharmaceuticalcompositions containing the same are expected to be administeredthroughout the lifetime of the subject being treated. Therefore, themode of administration of pharmaceutical compositions containing thecompounds should be such that will be easy and comfortable foradministration, preferably by self-administration, and such that willtake the smallest toll on the patient's wellbeing and course of life.

The repetitive and periodic administration of the compounds presentedherein or the pharmaceutical compositions containing the same can beeffected, for example, on a daily basis, i.e. once a day, morepreferably the administration is effected on a weekly basis, i.e. once aweek, more preferably the administration is effected on a monthly basis,i.e. once a month, and most preferably the administration is effectedonce every several months (e.g., every 1.5 months, 2 months, 3 months, 4months, 5 months, or even 6 months).

As discussed hereinabove, some of the limitations for using presentlyknown aminoglycosides as truncation mutation readthrough drugs areassociated with the fact that they are primarily antibacterial (used asantibiotic agents). Chronic use of any antibacterial agents is highlyunwarranted and even life threatening as it alters intestinal microbialflora which may cause or worsen other medical conditions such as flaringof inflammatory bowel disease, and may cause the emergence of resistancein some pathological strains of microorganisms.

In some embodiments, the compounds presented herein have substantiallyno antibacterial activity. By “no antibacterial activity” it is meantthat the minimal inhibition concentration (MIC) thereof for a particularstrain is much higher than the concentration of a compound that isconsidered an antibiotic with respect to this strain. Further, the MICof these compounds is notably higher than the concentration required forexerting truncation mutation suppression activity.

Being substantially non-bactericidal, the compounds presented herein donot exert the aforementioned adverse effects and hence can beadministered via absorption paths that may contain benign and/orbeneficial microorganisms that are not targeted and thus theirpreservation may even be required. This important characteristic of thecompounds presented herein renders these compounds particularlyeffective drugs against chronic conditions since they can beadministered repetitively and during life time, without causing anyantibacterial-related adverse, accumulating effects, and can further beadministered orally or rectally, i.e. via the GI tract, which is a veryhelpful and important characteristic for a drug directed at treatingchronic disorders.

As discussed hereinabove, according to some embodiments, the compoundspresented herein are selective towards the eukaryotic cellulartranslation system versus that of prokaryotic cells, namely thecompounds exhibit higher activity in eukaryotic cells, such as those ofmammalian (humans) as compared to their activity in prokaryotic cells,such as those of bacteria. Without being bound by any particular theory,it is assumed that the compounds presented herein, which are known toact by binding to the A-site of the 16S ribosomal RNA while the ribosomeis involved in translating a gene, have a higher affinity to theeukaryotic ribosomal A-site, or otherwise are selective towards theeukaryotic A-site, versus the prokaryotic ribosomal A-site, as well asthe mitochondrial ribosomal A-site which resembles its prokaryoticcounterpart.

As used herein the term “about” refers to ±10%.

The terms “comprises”, “comprising”, “includes”, “including”, “having”and their conjugates mean “including but not limited to”.

The term “consisting of” means “including and limited to”.

The term “consisting essentially of” means that the composition, methodor structure may include additional ingredients, steps and/or parts, butonly if the additional ingredients, steps and/or parts do not materiallyalter the basic and novel characteristics of the claimed composition,method or structure.

As used herein, the singular form “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof. Throughout this application,various embodiments of this invention may be presented in a rangeformat. It should be understood that the description in range format ismerely for convenience and brevity and should not be construed as aninflexible limitation on the scope of the invention. Accordingly, thedescription of a range should be considered to have specificallydisclosed all the possible subranges as well as individual numericalvalues within that range. For example, description of a range such asfrom 1 to 6 should be considered to have specifically disclosedsubranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4,from 2 to 6, from 3 to 6 etc., as well as individual numbers within thatrange, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of thebreadth of the range.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals therebetween.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

It is expected that during the life of a patent maturing from thisapplication many relevant aminoglycosides having a 5″-alkyl group willbe developed and the scope of this term is intended to include all suchnew technologies a priori.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Various embodiments and aspects of the present invention as delineatedhereinabove and as claimed in the claims section below find experimentalsupport in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions illustrate some embodiments of the invention in anon-limiting fashion.

Example 1 Chemical Synthesis

Synthetic Procedures:

Compounds NB118, NB119, NB122, NB123, NB124, NB125, NB127 and NB128 weresynthesized according to a general procedure that involves constructionof Ring III as two individual compounds possessing (S)-5-methyl and(R)-5-methyl with already established stereochemistry (Compound (S)-17and Compound (R)-18), and using them as donors for the glycosylationreactions. These donors were readily accessible from the knownthioglycoside Compound 7 as illustrated in Scheme 2 below (wherein “a”represents 1,1-dimethoxypropane, CSA, acetone, room temperature; “b”represents Dess-Martin periodinane (DMP), DCM, room temperature; “c”represents MeMgBr, THF, −30° C.; “d” represents TsCl, Py, 4-DMAP, roomtemperature; “e” represents NaN₃, HMPA, DMF, 70° C.; “f” representsacetic acid/water (8:2), reflux; “g” represents BzCl, Py, 4-DMAP, roomtemperature; “h” represents NBS, acetone/water (8:2), −30° C.; and “I”represents CCl3CN, DBU, DCM, 0° C.).

Selective protection of C2- and C3-hydroxyls by isopropylidine(2,2-dimethoxy propane/acetone, CSA) was followed by oxidation of theremaining primary alcohol using Dess-Martin periodinane (DMP,dichloromethane) to afford the aldehyde Compound 8 in 70% isolated yieldfor two steps. Treatment of Compound 8 with MeMgBr gave thecorresponding secondary alcohol as a mixture of C5-diasteromers (4:1ratio) in 88% isolated yield. This mixture was separated by flash columnchromatography and the major diastereomer was separately subjected forthe assignment of absolute stereochemistry at the C5-position (videinfra). This study established that the major and minor diastereomersexhibit (R)— and (S)-configuration, respectively (Compounds (R)-9 and(S)-10).

The following steps in Scheme 2 were separately performed on eachdiastereomer. Tosylation (TsCI, pyridine, 4-DMAP) of the secondaryalcohol was followed by S_(N)2 displacement of the correspondingtosylates (Compounds (R)-11 and (S)-12) with NaN₃ (DMF, HMPA) to furnishthe azides Compounds (S)-13 and (R)-14 with inverted configurations.Hydrolysis of the isopropylidene ketal with aqueous acetic acid,followed by benzoylation of the resulted secondary alcohols, providedthe benzoates Compounds (S)-15 and (R)-16. Earlier studies on theassembly of the pseudo-trisaccharides NB30 and NB54 have demonstratedthat the desired C5 acceptors are less reactive in glycosylationreactions, and trichloroacetimidate donors gave satisfactory results.

It is noted that the glycosylation reaction using thioglycoside donorssuch as (S)-15 and (R)-16 (see, Scheme 2 above) as donors, may beafforded in the presence of various glycosylation reagents includingN-iodosucinimide (NIS) and trifloromethane sulfonic acid (HOTf); or NISand silver triflate (AgOTf).

Therefore, the thioglycosides Compounds (S)-15 and (R)-16 were convertedto the corresponding trichloroacetimidates Compounds (S)-17 and (R)-18in two successive steps; hydrolysis with NBS in aqueous acetone andtreatment of the resulted hemiacetals with CC₁₃CN in the presence ofDBU. The donors Compounds (S)-17 and (R)-18 were used in glycosylationreactions without further purification.

The synthesis of the exemplary pseudo-trisaccharides compounds, NB118,NB119, NB122 and NB123, was accomplished from the correspondingselectively protected pseudo-disaccharide acceptors Compounds 19 and 20,as previously reported (WO 2007/113841), and the donors Compounds (S)-17and (R)-18, by using essentially the same chemical transformations, asillustrated in Scheme 3 below (wherein “a” represents BF₃.Et₂O, DCM, 4 ÅMS, −20° C.; “b” represents MeNH₂-EtOH, room temperature; and “c”represents PMe₃, NaOH, THF, room temperature).

Lewis acid (BF₃.Et₂O/DCM) promoted glycosylation furnished the protectedpseudo-trisaccharides Compounds 21-24 in 79-85% isolated yields,exclusively as beta-anomers at the newly generated glycosidic linkage.Two sequential deprotection steps: treatment with methylamine to removeall the ester protection, and the Staudinger reaction (Me₃P, THF/NaOH)to convert azides to corresponding amines, then afforded the targetcompounds NB118, NB119, NB122 and NB123 in 79-82% isolated yields fortwo steps.

The structures of all exemplary compounds NB118, NB119, NB122, NB123,NB124, NB125, NB127 and NB128 were confirmed by a combination of various1D and 2D NMR techniques, including 2D ¹H-¹³C HMQC and HMBC, 2D COSY,and 1D selective TOCSY experiments, along with mass spectral analysis(see ESM).

FIG. 1A-C present the synthesis plan of C5-diasteromeric esters (R,X)-27and (S,X)-28, reagents and conditions, wherein “a” represents DCC,4-DMAP, CSA, DCM, at room temperature (FIG. 1A); ¹H NMR spectra of(R,X)-27 and (S,X)-28, wherein the chemical shift differences (□⊏)between particular protons of (R,X)-27 and (S,X)-28 are highlighted(FIG. 1B); and assignment of absolute configuration at the 5-carbon(denoted by X) of the major alcohol Compound 9 by Sector rule (FIG. 1C).

For the assignment of the stereochemistry at the side-chain C5-alcoholsin Compounds 9 and 10 (see, Scheme 2), the major product Compound 9 wasseparately coupled (using DCC, 4-DMAP, CSA) with(R)-2-methoxy-2(1-naphthyl)propanoic acid (R)-MαNP and (S)-MαNP of knownabsolute stereochemistry, to afford the corresponding esters(R,X)-MαNP-27 and (S,X)-MαNP-28 (see, FIG. 1A), according to previouslyreported procedure. The absolute configuration at the C5-position(denoted by X) was then determined by using ¹H NMR anisotropy method(FIG. 1B-C): the chemical shift difference [Δδ=δ(R, X)−δ(S, X)] for H-3(−0.15) and H-4 (−0.30) was negative, while that for H-6 (+0.28) waspositive. An arrangement of the structures (R,X)-MαNP-27 and(S,X)-MαNP-28 according to the Sector rule (see, FIG. 1C: OMαNP and H-5are positioned on the front and back, respectively, while the Δδpositive part is on the right side of the MαNP plane and the Δδ negativepart is on the left side) then confirmed the R configuration (X=R) ofthe C5 in Compound 9.

Following similar synthetic procedures and synthetic rational, thesynthesis of the pseudo-trisaccharides NB124, NB125, NB127 and NB128 wasaccomplished (See, Scheme 4 below) from the corresponding selectivelyprotected pseudo-disaccharide acceptors Compounds 219 and 220, aspreviously reported (WO 2007/113841), and the donors Compounds (S)-17and (R)-18, by using essentially the same chemical transformations asillustrated in Scheme 3 (wherein “a” represents BF₃.Et₂O, DCM, 4 Å MS,−20° C.; “b” represents MeNH₂-EtOH, room temperature; and “c” representsPMe₃, NaOH, THF, room temperature).

Materials and Methods:

All reactions were carried out under an argon atmosphere with anhydroussolvents, unless otherwise noted.

All chemicals unless otherwise stated, were obtained from commercialsources such as Sigma-Aldrich, Fluka and the likes.

Reactions were monitored by TLC on Silica Gel 60 F₂₅₄ (0.25 mm, Merck),and spots were visualized by charring with a yellow solution containing(NH₄)Mo₇O₂₄.4H₂O (120 grams) and (NH₄)₂Ce(NO₃)₆ (5 grams) in 10% H₂SO₄(800 ml).

Column chromatography was performed on a Silica Gel 60 (70-230 mesh).

1D and 2D NMR spectra were routinely recorded on a Bruker Avance™ 500spectrometer.

Mass spectra analysis were obtained either on a Bruker Daltonix Apex 3mass spectrometer under electron spray ionization (ESI), or by a TSQ-70Bmass spectrometer (Finnigan Mat).

In all biological tests, all tested aminoglycosides were in theirsulfate salt forms. The concentrations reported refer to that of thefree amine form of each aminoglycoside.

Preparation of 4-Methylphenyl2,3-O-1-methylethylidene-1-thio-β-D-ribopentodialdo-1,4-furanoside(Compound 8)

A mixture of 4-methylphenyl 1-thio-β-D-ribofuranoside (Compound 7, 25grams, 0.097 mol) and 1,1-dimethoxypropane (22.3 ml, 0.39 mol) inacetone (500 ml) was stirred at room temperature for about five minutesand then catalytic amount of CSA (1.0 grams) and MgSO₄ (5.0 grams) wereadded. The reaction progress was monitored by TLC, which indicatedcompletion after 5 hours. The reaction mixture was diluted with ethylacetate and washed with saturated NaHCO₃ and brine. The combined organiclayer was dried over MgSO₄, evaporated and subjected to columnchromatography (EtOAc/Hexane) to afford the desired 2,3-isopropylidenederivative in 82% yield (23.5 grams).

¹H NMR (500 MHz, CDCl₃): δ_(H)3.73-3.85 (m, 2H, H-5), 4.37 (m, 1H, H-4),4.74 (dd, 1H, J₁=2.5, J₂=6.0 Hz, H-2), 4.80 (dd, 1H, J₁=1.7, J₂=6.0 Hz,H-3), 5.52 (d, 1H, J=2.5 Hz, H−1). Additional peaks in the spectrum wereidentified as follows: δ_(H)1.37 (s, 3H, isopropylidene-CH₃), 1.53 (s,3H, isopropylidene-CH₃), 2.35 (s, 3H, aryl-CH₃), 7.16 (d, 2H, J=8.0 Hz),7.42 (d, 2H, J=8.0 Hz).

¹³C NMR (125 MHz, CDCl₃): δc 21.0 (CH₃), 25.2 (CH₃), 26.8 (CH₃), 63.2(C-5), 81.8 (C-3), 85.7 (C-2), 87.7 (C-4), 93.0 (C-1), 113.3(quaternary-C), 129.2 (Ar), 129.9 (Ar), 132.3 (Ar), 138.0 (Ar).

MALDI TOFMS calculated for C₁₅H₂₀O₄SNa ([M+Na]⁺) m/e 319.1; measured m/e319.09.

The product the above step (22 grams, 0.074 mol) was stirred indichloromethane (500 ml) at room temperature to which Dess-Martinperiodinane (DMP, 34.6 grams, 0.082 mol) and MgSO₄ (5.0 grams) wereadded. The reaction progress was monitored by TLC, which indicatedcompletion after 8 hours. The reaction mixture was diluted with etherand washed with saturated NaHCO₃, Na₂S₂O₃, and brine. The combinedorganic layer was dried over MgSO₄, evaporated and subjected to columnchromatography (EtOAc/Hexane) to yield Compound 8 (18.0 grams, 85%yield).

¹H NMR (500 MHz, CDCl₃): δ_(H) 4.49 (s, 1H, H-4), 4.69 (d, 1H, J=6.5 Hz,H-2), 5.21 (d, 1H, J=6.0 Hz, H-3), 5.86 (s, 1H, H−1), 9.80 (s, 1H, H-5,CHO). Additional peaks in the spectrum were identified as follows: δ_(H)1.37 (s, 3H, isopropylidene-CH₃), 1.52 (s, 3H, isopropylidene-CH₃), 2.36(s, 3H, Ar—CH₃), 7.19 (d, 2H, J=8.0 Hz, Ar), 7.41 (d, 2H, J=8.0 Hz, Ar).

¹³C NMR (125 MHz, CDCl₃): δc 21.0 (CH₃), 25.1 (CH₃), 26.2 (CH₃), 87.1(C-3), 84.5 (C-2), 89.9 (C-4), 92.6 (C-1), 113.3 (quaternary-C), 128.9(Ar), 130.0 (Ar), 131.0 (Ar), 137.8 (Ar), 200.3 (CHO).

MALDI TOFMS calculated for C₁₅H₁₉O₄S ([M+H]⁺) m/e 295.1; measured m/e295.1.

Preparation of 4-Methylphenyl6-deoxy-2,3-O-1-methylethylidene-1-thio-β-D-allofuranoside (Compound(R)-9) and 4-methylphenyl6-deoxy-2,3-O-1-methylethylidene-1-thio-α-L-talofuranoside (Compound(S)-10)

The aldehyde Compound 8 (17 grams, 0.057 mol) was stirred in THF (200ml) at −30° C. for 30 minutes to which the solution of MeMgBr (1.4 M inTHF/Toluene, 235 ml, 0.171 mol) was added drop wise with syringe. Thereaction mixture was stirred for 2 hours at the same temperature andprogress was monitored by TLC. After completion, the reaction mixturewas quenched with saturated NH₄Cl and extracted with ethyl acetate. Thecombined organic layer was dried over MgSO₄ and evaporated. The crudeproduct was purified by column chromatography (EtOAc/Hexane) to afford4:1 ratio of two C5-diastereomers in 88% yield: the major productCompound (5R)-9 (13 grams, R_(f)=0.38 in EtOAc/Hexane 1:4) and the minorproduct Compound (5 S)-10 (3 grams, R_(f)=0.48 in EtOAc/Hexane 1:4). Theabsolute configuration at the C5-position was determined by using ¹H NMRanisotropy method as described below.

Data for Compound (5R)-9:

[α]_(D) ²⁰=−191.4 (c=1.02, CHCl₃). ¹H NMR (500 MHz, CDCl₃): δ_(H) 1.25(d, 3H, J=6.3 Hz, CH₃), 4.06 (m, 2H, H-4 and H-5), 4.68 (dd, 1H, J₁=2.8,J₂=6.3 Hz, H-2), 4.87 (t, 1H, J=5.0 Hz, H-3), 5.46 (d, 1H, J=2.8 Hz,H−1). Additional peaks in the spectrum were identified as follows: δ_(H)1.37 (s, 3H, isopropylidene-CH₃), 1.53 (s, 3H, isopropylidene-CH₃), 2.34(s, 3H, Ar—CH₃), 7.15 (d, 2H, J=8.0 Hz, Ar), 7.42 (d, 2H, J=8.0 Hz, Ar).

¹³CNMR (125 MHz, CDCl₃): δc 18.5 (C-6), 21.0 (CH₃), 25.2 (CH₃), 26.9(CH₃), 67.3 (C-5), 80.2 (C-3), 85.4 (C-2), 91.4 (C-4), 92.5 (C-1), 113.4(quaternary-C), 129.2 (Ar), 129.8 (Ar), 132.3 (Ar), 137.9 (Ar).

MALDI TOFMS calculated for C₁₆H₂₂O₄SNa ([M+Na]⁺) m/e 333.1; measured m/e333.1.

Data for Compound (5S)-10:

[α]_(D) ²⁰=−199.7 (c=1.04, CHCl₃). ¹H NMR (500 MHz, CDCl₃): δ_(H) 1.27(d, 3H, J=6.3 Hz, CH₃), 3.90 (m, 1H, H-5), 4.08 (dd, 1H, J₁=1.3, J₂=5.6Hz, H-4), 4.71 (dd, 1H, J₁=1.3, J₂=6.0 Hz, H-3), 4.76 (dd, 1H, J₁=2.1,J₂=6.0 Hz, H-2), 5.57 (d, 1H, J=2.0 Hz, H-1). Additional peaks in thespectrum were identified as follows: δ_(H) 1.36 (s, 3H,isopropylidene-CH₃), 1.54 (s, 3H, isopropylidene-CH₃), 2.35 (s, 3H,Ar—CH₃), 7.17 (d, 2H, J=8.0 Hz, Ar), 7.43 (d, 2H, J=8.0 Hz, Ar).

¹³C NMR (125 MHz, CDCl₃): δc 19.2 (C-6), 21.0 (CH₃), 25.2 (CH₃), 26.8(CH₃), 67.9 (C-5), 82.4 (C-3), 85.7 (C-2), 91.6 (C-4), 93.0 (C-1), 113.3(quaternary-C), 129.4 (Ar), 129.9 (Ar), 131.9 (Ar), 137.9 (Ar).

MALDI TOFMS calculated for C₁₆H₂₂O₄SNa ([M+Na]⁺) m/e 333.1; measured m/e333.1.

Preparation of esters Compound (R,X)-27 and Compound (S,X)-28 for theAssignment of Absolute Configuration at C5

A mixture of (R)-2-methoxy-2(1-naphthyl)propanoic acid [(R)-MαNP] or(S)-MαNP (0.07 grams, 0.0003 mol), 4-dimethylaminopyridine (DMAP, 0.05grams, 0.0004 mol), 10-camphorsulfonic acid (CSA, 0.025 grams), and1,3-dicyclohexylcarbodiimide (DCC, 0.240 grams, 0.0016 mol) was stirredin CH₂Cl₂ (30 ml) at 0° C. The major alcohol 9 from the above (0.1grams, 0.0003 mol), was dissolved in CH₂Cl₂ (5 ml), slowly added to theabove stirred mixture, and the reaction was left at room temperature forovernight. The mixture was diluted with EtOAc and washed with 1% HClsolution, saturated NaHCO₃ and brine. The combined organic layer wasdried over MgSO₄, evaporated and subjected to a column chromatography(EtOAc/Hexane) to yield the desired esters Compound (R,X)-27 (0.135grams, 80%) or Compound (S,X)-28 (0.138 grams, 80%).

Data for Compound (R,X)-27:

¹H NMR (500 MHz, CDCl₃): δ_(H) 1.23 (d, 3H, J=6.3 Hz, CH₃), 3.54 (d, 1H,J=6.1 Hz, H-3), 3.72 (d, 1H, J=9.0 Hz, H-4), 4.18 (dd, 1H, J₁=2.3,J₂=6.1 Hz, H-2), 5.08 (m, 1H, H−5), 5.32 (d, 1H, J=2.4 Hz, H−1).Additional peaks in the spectrum were identified as follows: δ_(H) 1.00(s, 3H, isopropylidene-CH₃), 1.32 (s, 3H, isopropylidene-CH₃), 2.04 (s,3H, CH₃), 2.32 (s, 3H, Ar—CH₃), 3.14 (s, 3H, OCH₃), 7.11 (d, 2H, J=8.0Hz, Ar), 7.28-7.31 (m, 2H, Ar), 7.48-7.56 (m, 3H, Ar), 7.65 (d, 1H,J=8.0 Hz, Ar), 7.85 (dd, 2H, J₁=4.7, J₂=8.0 Hz, Ar), 8.47 (d, 1H, J=8.0Hz, Ar).

¹³C NMR (125 MHz, CDCl₃): δc 17.1 (C-6), 21.0 (CH₃), 21.5 (CH₃), 24.8(CH₃), 26.6 (CH₃), 50.9 (OCH₃), 70.5 (C-5), 81.2 (C-3), 81.3(quaternary-C), 84.8 (C-2), 88.0 (C-4), 92.5 (C-1), 112.9(quaternary-C), 124.7 (Ar), 125.0 (Ar), 125.7 (Ar), 125.8 (Ar), 126.6(Ar), 128.8 (Ar), 129.5 (Ar), 129.7 (Ar), 130.3 (Ar), 131.2 (Ar), 131.3(Ar), 134.0 (Ar), 134.6 (Ar), 137.2 (Ar), 173.1 (C═O).

MALDI TOFMS calculated for C₃₀H₃₄O₆SNa ([M+Na]⁺) m/e 545.2; measured m/e545.2.

Data for Compound (S,X)-28:

¹H NMR (500 MHz, CDCl₃): δc 0.95 (d, 3H, J=6.3 Hz, CH₃), 3.84 (dd, 1H,J₁=1.5, J₂=6.2 Hz, H−3), 3.87 (dd, 1H, J₁=1.5 and J₂=6.2 Hz, H−4), 4.08(dd, 1H, J₁=3.4, J₂=6.1 Hz, H−2), 5.06 (m, 1H, H−5), 5.27 (d, 1H, J=3.4Hz, H−1). Additional peaks in the spectrum were identified as follows:δ_(H) 1.14 (s, 3H, isopropylidene-CH₃), 1.41 (s, 3H,isopropylidene-CH₃), 2.09 (s, 3H, CH₃), 2.33 (s, 3H, Ar—CH₃), 3.14 (s,3H, OCH₃), 7.12 (d, 2H, J=8.0 Hz, Ar), 7.35 (d, 2H, J=8.0 Hz, Ar),7.49-7.67 (m, 3H, Ar), 7.69 (d, 1H, J=8.0 Hz, Ar), 7.88 (d, 2H, J=8.0Hz, Ar), 8.41 (d, 1H, J=8.0 Hz, Ar).

¹³C NMR (125 MHz, CDCl₃): δc 16.0 (C-6), 21.0 (CH₃), 21.5 (CH₃), 24.9(CH₃), 26.8 (CH₃), 50.8 (OCH₃), 71.4 (C-5), 81.0 (C-3), 81.5(quaternary-C), 84.7 (C-2), 87.5 (C-4), 92.6 (C-1), 113.3(quaternary-C), 124.7 (Ar), 125.2 (Ar), 125.7 (Ar), 126.0 (Ar), 126.4(Ar), 128.6 (Ar), 129.4 (Ar), 129.7 (Ar), 130.3 (Ar), 131.3 (Ar), 131.5(Ar), 133.8 (Ar), 134.8 (Ar), 137.4 (Ar), 173.4 (C═O).

MALDI TOFMS calculated for C₃₀H₃₄O₆SNa ([M+Na]⁺) m/e 545.2; measured mle545.2.

Preparation of 4-Methylphenyl6-deoxy-5-O-tosyl-2,3-O-1-methylethylidene-1-thio-β-D-allofuranoside(Compound (R)-11)

To a stirred solution of Compound (R)-9 (13 grams, 0.041 mol) inpyridine (200 ml) at 0° C., were added tosyl chloride (15.6 grams, 0.082mol) and 4-DMAP (1 gram). The reaction temperature was raised to roomtemperature and progress was monitored by TLC. After completion (36hours), the reaction mixture was diluted with ethyl acetate andsequentially washed with 1% aqueous HCl solution, saturated NaHCO₃, andbrine. The combined organic layer was dried over MgSO₄, evaporated andsubjected to column chromatography (EtOAc/Hexane) to obtain Compound(R)-11 (16.0 grams) in 82% yield.

¹H NMR (500 MHz, CDCl₃): δ_(H) 1.28 (d, 3H, J=6.2 Hz, CH₃), 3.99 (d, 1H,J=8.6 Hz, H−4), 4.60 (dd, 1H, J₁=2.0, J₂=6.2 Hz, H−2), 4.67 (d, 1H,J=6.2 Hz, H−3), 4.92 (m, 1H, H−5), 5.48 (d, 1H, J=1.8 Hz, H−1).Additional peaks in the spectrum were identified as follows: δ_(H) 1.30(s, 3H, isopropylidene-CH₃), 1.48 (s, 3H, isopropylidene-CH₃), 2.34 (s,3H, Ar—CH₃), 2.45 (s, 3H, Ar—CH₃) 7.13 (d, 2H, J=8.0 Hz, Ar), 7.30-7.38(m, 4H, Ar), 7.87 (d, 2H, J=8.0 Hz, Ar).

¹³C NMR (125 MHz, CDCl₃): δc 18.0 (C-6), 21.0 (CH₃), 21.6 (CH₃), 25.0(CH₃), 26.6 (CH₃), 77.1 (C-5), 81.2 (C-3), 85.0 (C-2), 87.9 (C-4), 92.3(C-1), 113.6 (quaternary-C), 127.9 (Ar), 129.8 (2C, Ar), 129.9 (Ar),131.0 (Ar), 133.8 (Ar), 137.4 (Ar), 144.8 (Ar).

MALDI TOFMS calculated for C₂₃H₂₉O₆S₂ ([M+H]⁺) mle 465.1; measured m/e465.1.

Preparation of 4-Methylphenyl6-deoxy-5-O-tosyl-2,3-O-1-methylethylidene-1-thio-α-L-alofuranoside(Compound (S)-12)

To a stirred solution of Compound (S)-10 (10 grams, 0.032 mol) inpyridine (200 ml) at 0° C., were added tosyl chloride (15.6 grams, 0.082mol) and 4-DMAP (1 gram). The reaction temperature was raised to roomtemperature and progress was monitored by TLC. After completion (36hours), the reaction mixture was diluted with ethyl acetate andsequentially washed with 1% aqueous HCl solution, saturated NaHCO₃, andbrine. The combined organic layer was dried over MgSO₄, evaporated andsubjected to column chromatography (EtOAc/Hexane) to obtain Compound(S)-12 (14.0 grams) in 88% yield.

¹HNMR (500 MHz, CDCl₃): δ_(H) 1.37 (d, 3H, J=6.4 Hz, CH₃), 4.09 (dd, 1H,J₁=2.8, J₂=4.3 Hz, H−4), 4.48 (dd, 1H, J₁=2.8, J₂=6.2 Hz, H−3), 4.55(dd, 1H, J₁=4.0, J₂=6.2 Hz, H−2), 4.82 (m, 1H, H−5), 5.25 (d, 1H, J=4.0Hz, H−1). Additional peaks in the spectrum were identified as follows:δ_(H) 1.30 (s, 3H, isopropylidene-CH₃), 1.50 (s, 3H,isopropylidene-CH₃), 2.35 (s, 3H, Ar—CH₃), 2.43 (s, 3H, Ar—CH₃) 7.12 (d,2H, J=8.0 Hz, Ar), 7.32 (d, 2H, J=8.0 Hz, Ar), 7.38 (d, 2H, J=8.0 Hz,Ar), 7.87 (d, 2H, J=8.0 Hz, Ar).

¹³CNMR (125 MHz, CDCl₃): δc 17.0 (C-6), 21.0 (Ar—CH₃), 21.6 (Ar—CH₃),25.3 (isopropylidene —CH₃), 27.2 (isopropylidene-CH₃), 78.3 (C-5), 81.2(C-3), 84.6 (C-2), 86.7 (C-4), 92.2 (C-1), 114.1 (quaternary-C), 127.7(Ar), 129.6 (Ar), 129.7 (Ar), 129.8 (Ar), 132.3 (Ar), 134.1 (Ar), 137.6(Ar), 144.7 (Ar).

MALDI TOFMS calculated for C₂₃H₂₈O₆S₂Na ([M+Na]⁺) m/e: 487.1; measuredm/e: 487.1

Preparation of 4-Methylphenyl5-azido-5,6-dideoxy-2,3-O-1-methylethylidene-1-thio-α-L-talofuranoside(Compound (S)-13)

To a stirred solution of Compound (R)-11 (15 grams, 0.032 mol) in DMF(250 ml) were added NaN₃ (10 grams, 0.15 mol) and HMPA (15 ml) at roomtemperature. The reaction temperature was raised to 70° C. and progresswas monitored by TLC. After completion (10 hours), the reaction mixturewas diluted with ethyl acetate and sequentially washed with 1% aqueousHCl solution, saturated NaHCO₃, and brine. The combined organic layerwas dried over MgSO₄, evaporated and subjected to column chromatography(EtOAc/Hexane) to obtain Compound (S)-13 (6 grams) in 55% yield.

¹HNMR (500 MHz, CDCl₃): δ_(H) 1.35 (d, 3H, J=6.2 Hz, CH₃), 3.73 (m, 1H,H−5), 3.99 (dd, 1H, J₁=3.0, J₂=6.7 Hz, H−4), 4.56 (dd, 1H, J₁=3.0,J₂=6.5 Hz, H−3), 4.70 (dd, 1H, J₁=2.0, J₂=6.2 Hz, H−2), 5.39 (d, 1H,J=3.2 Hz, H−1). Additional peaks in the spectrum were identified asfollows: δ_(H) 1.36 (s, 3H, isopropylidene-CH₃), 1.53 (s, 3H,isopropylidene-CH₃), 2.36 (s, 3H, Ar—CH₃), 7.15 (d, 2H, J=8.0 Hz, Ar),7.46 (d, 2H, J=8.0 Hz, Ar).

¹³CNMR (125 MHz, CDCl₃): δc 15.5 (C-6), 21.1 (Ar—CH₃), 25.4(isopropylidene-CH₃), 27.1 (isopropylidene-CH₃), 58.2 (C-5), 81.9 (C-3),85.1 (C-2), 88.9 (C-4), 91.9 (C-1), 114.2 (quaternary-C), 129.5 (Ar),129.7 (Ar), 132.4 (Ar), 138.8 (Ar).

MALDI TOFMS calculated for C₁₆H₂₀N₃O₃S ([M−H]⁻) mle: 334.1; measuredm/e: 334.1.

Preparation of 4-Methylphenyl5-azido-5,6-dideoxy-2,3-O-1-methylethylidene-1-thio-β-D-allofuranoside(Compound (R)-14)

To a stirred solution of Compound (S)-12 (13 grams, 0.028 mol) in DMF(250 ml) were added NaN₃ (10 grams, 0.15 mol) and HMPA (13 ml) at roomtemperature. The reaction temperature was raised to 70° C. and progresswas monitored by TLC. After completion (10 hours), the reaction mixturewas diluted with ethyl acetate and sequentially washed with 1% aqueousHCl solution, saturated NaHCO₃, and brine. The combined organic layerwas dried over MgSO₄, evaporated and subjected to column chromatography(EtOAc/Hexane) to obtain Compound (R)-14 (9 grams) in 97% yield.

¹HNMR (500 MHz, CDCl₃): δ_(H) 1.32 (d, 3H, J=6.2 Hz, CH₃), 3.81 (m, 1H,H−5), 3.89 (dd, 1H, J₁=2.1, J₂=8.3 Hz, H−4), 4.72 (dd, 1H, J₁=2.5,J₂=6.3 Hz, H−2), 4.77 (dd, 1H, J₁=2.1, J₂=6.3 Hz, H−3), 5.49 (d, 1H,J=2.5 Hz, H−1). Additional peaks in the spectrum were identified asfollows: δ_(H) 1.37 (s, 3H, isopropylidene-CH₃), 1.53 (s, 3H,isopropylidene-CH₃), 2.35 (s, 3H, Ar—CH₃), 7.15 (d, 2H, J=8.0 Hz, Ar),7.74 (d, 2H, J=8.0 Hz, Ar).

¹³CNMR (125 MHz, CDCl₃): δc 16.2 (C-6), 21.0 (Ar—CH₃), 25.2(isopropylidene-CH₃), 26.1 (isopropylidene-CH₃), 58.1 (C-5), 81.9 (C-3),85.1 (C-2), 89.1 (C-4), 92.2 (C-1), 113.8 (quaternary-C), 129.7 (Ar),129.8 (Ar), 131.6 (Ar), 137.6 (Ar).

MALDI TOFMS calculated for C₁₆H₂₀N₃O₃S ([M−H]⁻) m/e: 334.1; measuredm/e: 334.1.

Preparation of 4-Methylphenyl5-azido-5,6-dideoxy-2,3-O-dibenzoyl-1-thio-α-L-talofuranoside (Compound(S)-15)

Compound (S)-13 (6 grams, 0.018 mol) was stirred in a mixture of aceticacid-water (100 ml, 8:2) at 70° C. for over night. The reaction progresswas monitored by TLC, after completion, the reaction mixture was dilutedwith ethyl acetate and washed with saturated NaHCO₃ and brine. Thecombined organic layer was dried over MgSO₄, evaporated and subjected tocolumn chromatography (EtOAc/Hexane) to obtain desired isopropylidenedeprotected product (5 grams) in 96% yield.

¹HNMR (500 MHz, CDCl₃): δ_(H) 1.36 (d, 3H, J=6.2 Hz, CH₃), 3.61 (m, 1H,H−5), 3.82 (t, 1H, J=4.8 Hz, H−4), 4.13 (m, 2H, H−3 and H−2), 5.18 (d,1H, J=3.7 Hz, H−1). Additional peaks in the spectrum were identified asfollows: δ_(H) 2.35 (s, 3H, Ar—CH₃), 7.15 (d, 2H, J=8.0 Hz, Ar), 7.45(d, 2H, J=8.0 Hz, Ar).

¹³CNMR (125 MHz, CDCl₃): δc 15.2 (C-6), 21.0 (Ar—CH₃), 58.2 (C-5), 72.0(C-3), 74.9 (C-2), 86.5 (C-4), 90.5 (C-1), 128.8 (Ar), 129.7 (Ar), 133.0(Ar), 138.1 (Ar).

MALDI TOFMS calculated for C₁₃H₁₆N₃O₃S ([M−H]⁻) m/e: 294.1; measuredm/e: 294.08.

The product of the above step was stirred in pyridine (200 ml) at 0° C.to which BzCI (7.14 grams, 0.051) and 4-DMAP (1 gram) was added slowly.The reaction temperature was raised to room temperature and stirred forovernight. The reaction progress was monitored by TLC, after completion,reaction mixture was diluted with ethyl acetate and washed with 1% HClsolution, saturated NaHCO₃ and brine. The combined organic layer wasdried over MgSO₄, evaporated and subjected to column chromatography(EtOAc/Hexane) to obtain Compound (S)-15 (8.0 grams) in 94% yield.

¹HNMR (500 MHz, CDCl₃): δ_(H) 1.38 (d, 3H, J=6.2 Hz, CH₃), 3.81 (m, 1H,H−5), 4.22 (m, 1H, H−4), 5.55 (m, 1H, H−1), 5.56-5.58 (m, 2H, H−2 andH−3). Additional peaks in the spectrum were identified as follows: δ_(H)2.37 (s, 3H, Ar—CH₃), 7.21 (d, 2H, J=8.0 Hz, Ar), 7.34-7.42 (m, 4H, Ar),7.53-7.59 (m, 4H, Ar), 7.90 (dd, 2H, J₁=1.2, J₂=8.0 Hz, Ar), 7.99 (dd,2H, J₁=1.2, J₂=8.0 Hz, Ar).

¹³CNMR (125 MHz, CDCl₃): δc 15.2 (C-6), 21.1 (Ar—CH₃), 57.9 (C-5), 72.6(C-3), 74.4 (C-2), 85.1 (C-4), 88.4 (C-1), 127.8 (Ar), 128.3 (2C, Ar),128.9 (Ar), 129.0 (Ar), 129.6 (Ar), 129.7 (Ar), 129.8 (Ar), 133.4 (2C,Ar), 133.9 (Ar), 138.6 (Ar), 164.9 (C═O), 165.2 (C═O).

MALDI TOFMS calculated for C₂₇H₂₅N₃O₅SNa ([M+Na]⁺) m/e: 526.2; measuredm/e: 526.2.

Preparation of 4-Methylphenyl5-azido-5,6-dideoxy-2,3-O-dibenzoyl-1-thio-β-D-allofuranoside (Compound(R)-16)

Compound (R)-14 (8 grams, 0.023 mol) was stirred in a mixture of aceticacid-water (100 ml, 8:2) at 70° C. for over night. The reaction progresswas monitored by TLC, after completion, the reaction mixture was dilutedwith ethyl acetate and washed with saturated NaHCO₃ and brine. Thecombined organic layer was dried over MgSO₄, evaporated and subjected tocolumn chromatography (EtOAc/Hexane) to obtain the desiredisopropylidene deprotected product (6.5 grams) in 92% yield.

¹HNMR (500 MHz, CDCl₃): δ_(H) 1.36 (d, 3H, J=6.2 Hz, CH₃), 3.65 (m, 1H,H−5), 3.78 (dd, 1H, J₁=2.5, J₂=7.5 Hz, H−4), 4.09 (t, 1H, J=5.0 Hz,H−2), 4.15 (t, 1H, J=4.5 Hz, H−3), 5.18 (d, 1H, J=5.0 Hz, H−1).Additional peaks in the spectrum were identified as follows: δ_(H) 2.36(s, 3H, Ar—CH₃), 7.15 (d, 2H, J=8.0 Hz, Ar), 7.44 (d, 2H, J=8.0 Hz, Ar).

¹³CNMR (125 MHz, CDCl₃): δc 16.0 (C-6), 21.0 (Ar—CH₃), 59.0 (C-5), 71.9(C-3), 74.9 (C-2), 86.4 (C-4), 90.3 (C-1), 128.9 (Ar), 129.7 (Ar), 132.8(Ar), 138.1 (Ar).

MALDI TOFMS calculated for C₁₃H₁₆N₃O₃S ([M−H]⁻) m/e: 294.1; measuredm/e: 294.08.

The product from the above step was stirred in pyridine (200 ml) at 0°C. to which BzCI (7.14 grams, 0.051) and 4-DMAP (1 gram) was addedslowly. The reaction temperature was raised to room temperature andstirred for overnight. The reaction progress was monitored by TLC, aftercompletion, reaction mixture was diluted with ethyl acetate and washedwith 1% HCl solution, saturated NaHCO₃ and brine. The combined organiclayer was dried over MgSO₄, evaporated and subjected to columnchromatography (EtOAc/Hexane) to obtain Compound (R)-16 (9.5 grams) in93% yield.

¹HNMR (500 MHz, CDCl₃): δ_(H) 1.42 (d, 3H, J=6.7 Hz, CH₃), 3.74 (m, 1H,H−5), 4.24 (t, 1H, J=4.7 Hz, H−4), 5.53 (d, 1H, J=5.6 Hz, H−1), 5.50 (t,1H, J=5.5 Hz, H−2), 5.65 (t, 1H, J=5.5 Hz, H−3). Additional peaks in thespectrum were identified as follows: δ_(H) 2.38 (s, 3H, Ar—CH₃), 7.20(d, 2H, J=8.0 Hz, Ar), 7.38 (t, 4H, J=7.6 Hz, Ar), 7.51 (d, 2H, J=8.0Hz, Ar), 7.55 (t, 2H, J=8.0 Hz, Ar), 7.93-7.96 (m, 4H, Ar).

¹³CNMR (125 MHz, CDCl₃): δc 15.5 (C-6), 21.1 (Ar—CH₃), 58.5 (C-5), 71.8(C-3), 74.2 (C-2), 84.9 (C-4), 88.2 (C-1), 127.8 (Ar), 128.3 (2C, Ar),128.9 (2C, Ar), 129.6 (Ar), 129.7 (Ar), 129.8 (Ar), 133.3 (2C, Ar),133.8 (Ar), 138.6 (Ar), 164.9 (C═O), 165.0 (C═O).

MALDI TOFMS calculated for C₂₇H₂₅N₃O₅SNa ([M+Na]⁺) mle: 526.2; measuredm/e: 526.2

Preparation of L-Talofuranose, 5-azido-5,6-dideoxy-2,3-dibenzoate1-(2,2,2-trichloroethanimidate) (Compound (S)-17)

Compound (S)-15 (8 grams, 0.016 mol) was stirred in a mixture ofacetone-water (100 ml, 9:1) mixture at −30° C. for 10 minutes to whichN-bromosuccinimide (9.16 grams, 0.051 mol) was added slowly. Thereaction mixture was stirred at same temperature and the progress wasmonitored by TLC. After completion (3 hours), reaction mixture wasdiluted with ethyl acetate and washed saturated NaHCO₃, saturatedNa₂S₂O₃ and brine. The combined organic layer was dried over MgSO₄,evaporated to obtain 6.3 grams of corresponding hem iacetal. The hemiacetal was stirred in a mixture of dichloromethane (40 ml) andtrichloroacetonitrile (5 ml) at 0° C. for 10 minutes to which catalyticamount of DBU (0.3 ml) was added. The reaction mixture was stirred insame temperature and the progress was monitored by TLC. After completion(3 hours), the reaction mixture was diluted with DCM and washed withsaturated NH₄Cl. The combined organic layer was dried over MgSO₄ andconcentrated to obtain Compound (S)-17 (9 grams). The crude product wasdirectly used for the glycosylation reaction without purification.

Preparation of D-Allofuranose, 5-azido-5,6-dideoxy-2,3-dibenzoate1-(2,2,2-trichloroethanimidate) (Compound (R)-18)

Compound (R)-16 (9 grams, 0.018 mol) was stirred in a mixture ofacetone-water (100 ml, 9:1) mixture at −30° C. for 10 minutes to whichN-bromosuccinimide (9.0 grams, 0.050 mol) was added slowly. The reactionmixture was stirred at same temperature and the progress was monitoredby TLC. After completion (3 hours), reaction mixture was diluted withethyl acetate and washed saturated NaHCO₃, saturated Na₂S₂O₃ and brine.The combined organic layer was dried over MgSO₄, evaporated to obtain6.5 grams of corresponding hemiacetal. The hemiacetal was stirred in amixture of dichloromethane (50 ml) and trichloroacetonitrile (6 ml) at0° C. for 10 minutes to which catalytic amount of DBU (0.3 ml) wasadded. The reaction mixture was stirred in same temperature and theprogress was monitored by TLC. After completion (3 hours), the reactionmixture was diluted with DCM and washed with saturated NH₄Cl. Thecombined organic layer was dried over MgSO₄ and concentrated to obtainCompound (R)-18 (9 grams). The crude product was directly used for theglycosylation reaction without purification.

Preparation of5-O-(5-Azido-5,6-dideoxy-2,3-O-dibenzoyi-α-L-talofuranosyl)-3,4′,6′,6-tetra-O-acetyl-2,1,3-triazidoparomamine (Compound (S)-21)

Anhydrous CH₂Cl₂ (15 ml) was added to a powdered, flame-dried 4 Åmolecular sieves (2.0 grams), followed by the addition acceptor Compound19 (0.75 grams, 0.0013 mol) and donor Compound (S)-17 (2.1 grams, 0.0039mol). The reaction mixture was stirred for 10 min at room temperatureand was then cooled to −20° C. A catalytic amount of BF3-Et₂O (0.1 ml)was added and the mixture was stirred at −15° C. and the reactionprogress was monitored by TLC, which indicated the completion after 60minutes. The reaction mixture was diluted with ethyl acetate and washedwith saturated NaHCO₃ and brine. The combined organic layer was driedover MgSO₄, evaporated and subjected to column chromatography(EtOAc/Hexane) to obtain Compound (S)-21 (1.0 grams) in 80% yield.

¹HNMR (500 MHz, CDCl₃): “Ring I” δ_(H) 3.65 (dd, 1H, J₁=4.2, J₂=9.7 Hz,H−2′), 4.20 (d, 1H, J=11.1 Hz, H−6′), 4.26 (dd, 1H, J₁=3.1, J₂=12.6 Hz,H−6′), 4.54 (m, 1H, H−5′), 5.08 (dd, 1H, J₁=9.3, J₂=10.7 Hz, H−4′), 5.41(t, 1H, J=9.9 Hz, H−3′), 5.85 (d, 1H, J=3.7 Hz, H−1′); “Ring II” δ_(H)1.64 (ddd, 1H, J₁=J₂=J₃=12.5 Hz, H−2_(ax)), 2.42 (td, 1H, J₁=4.5,J₂=12.5 Hz, H−2_(eq)), 3.49-3.56 (m, 2H, H−1 and H−3), 3.74 (t, 1H,J=9.5 Hz, H−4), 3.87 (t, 1H, J=8.7 Hz, H−5), 5.02 (d, 1H, J=10.1 Hz,H−6); “Ring III” δ_(H) 1.27 (d, 3H, J=6.9 Hz, CH₃), 3.72 (m, 1H, H−5″),4.35 (t, 1H, J=6.6 Hz, H−4″), 5.43 (dd, 1H, J₁=5.1, J₂=7.4 Hz, H−3″),5.62 (d, 1H, J=3.8 Hz, H−2″), 5.66 (s, 1H, H−1″). Additional peaks inthe spectrum were identified as follows: δ_(H) 2.04 (s, 3H, OAc), 2.10(s, 3H, OAc), 2.11 (s, 3H, OAc), 2.23 (s, 3H, OAc), 7.35-7.43 (m, 4H,Ar), 7.53-7.60 (m, 2H, Ar), 7.89-7.95 (m, 4H, Ar).

¹³CNMR (125 MHz, CDCl₃): δc 15.3 (C-6″), 20.5 (OAc), 20.6 (2C, OAc),20.9 (OAc), 31.6 (C-1), 58.3, 58.5, 59.3, 61.7, 61.8, 68.0, 68.2, 70.9,71.8, 73.6, 74.6, 78.1, 79.5, 84.4, 96.6 (C-1′), 107.6 (C-1″), 128.4(Ar), 128.5 (2C, Ar), 128.7 (Ar), 129.6 (2C, Ar), 133.5 (Ar), 133.6(Ar), 164.8 (C═O), 165.3 (C═O), 169.7 (C═O), 169.9 (C═O), 170.1 (C═O),170.6 (C═O).

MALDI TOFMS calculated for C₄₀H₄₃N₁₂O₁₆ ([M−H]⁻) m/e: 947.3; measuredm/e: 947.28.

Preparation of5-O-(5-Azido-5,6-dideoxy-2,3-O-dibenzoyl-β-D-allofuranosyl)-3′,4′,6′,6-tetra-O-acetyl-2′,1,3-triazidoparomamine (Compound (R)-22)

Anhydrous CH₂Cl₂ (15 ml) was added to a powdered, flame-dried 4 Åmolecular sieves (2.0 grams), followed by the addition acceptor Compound19 (0.75 grams, 0.0013 mol) and donor Compound (R)-18 (2.1 grams, 0.0039mol). The reaction mixture was stirred for 10 minutes at roomtemperature and was then cooled to −20° C. A catalytic amount ofBF₃-Et₂O (0.1 ml) was added and the mixture was stirred at −15° C. andthe reaction progress was monitored by TLC, which indicated thecompletion after 60 minutes. The reaction mixture was diluted with ethylacetate and washed with saturated NaHCO₃ and brine. The combined organiclayer was dried over MgSO₄, evaporated and subjected to columnchromatography (EtOAc/Hexane) to obtain Compound (R)-22 (1.02 grams) in82% yield.

¹HNMR (500 MHz, CDCl₃): “Ring I” δ_(H) 3.55 (dd, 1H, J₁=4.5 and J₂=10.7Hz, H−2′), 4.17 (d, 1H, J=13.1 Hz, H−6′), 4.30 (dd, 1H, J₁=4.2 andJ₂=12.4 Hz, H−6′), 4.56 (m, 1H, H−5′), 5.08 (t, 1H, J=9.7 Hz, H−4′),5.43 (t, 1H, J=9.9 Hz, H−3′), 5.83 (d, 1H, J=3.9 Hz, H−1′); “Ring II”δ_(H) 1.64 (ddd, 1H, J₁=J₂=J₃=12.5 Hz, H−2_(ax)), 2.42 (td, 1H, J₁=4.5and J₂=12.5 Hz, H−2_(eq)), 3.49-3.56 (m, 2H, H−1 and H−3), 3.74 (t, 1H,J=10.0 Hz, H−4), 3.92 (t, 1H, J=9.1 Hz, H−5), 5.03 (d, 1H, J=9.9 Hz,H−6); “Ring III” δ_(H) 1.41 (d, 3H, J=6.9 Hz, CH₃), 3.76 (m, 1H, H−5″),4.39 (t, 1H, J=4.9 Hz, H−4″), 5.50 (dd, 1H, J₁=5.1 and J₂=7.0 Hz, H−3″),5.60 (d, 1H, J=4.9 Hz, H−2″), 5.68 (s, 1H, H−1″). Additional peaks inthe spectrum were identified as follows: δ_(H) 2.06 (s, 3H, OAc), 2.09(s, 3H, OAc), 2.11 (s, 3H, OAc), 2.34 (s, 3H, OAc), 7.37-7.41 (m, 4H,Ar), 7.57 (m, 2H, Ar), 7.92 (d, 4H, J=8.0 Hz Ar).

¹³CNMR (125 MHz, CDCl₃): δ_(H) 15.1 (C-6″), 20.5 (OAc), 20.6 (OAc), 20.7(OAc), 20.8 (OAc), 31.7 (C-1), 58.2 (2C), 58.6, 61.7 (2C), 68.0, 68.1,70.7, 71.4, 73.7, 74.6, 77.8, 79.2, 83.9, 96.6 (C-1′), 107.1 (C-1″),128.4 (2C, Ar), 128.7 (Ar), 128.8 (Ar), 129.6 (2C, Ar), 133.4 (Ar),133.5 (Ar), 164.9 (C═O), 165.4 (C═O), 169.7 (2C, C═O), 169.9 (C═O),170.6 (C═O).

MALDI TOFMS calculated for C₄₀H₄₄N₁₂O₁₆ Na ([M+Na]⁺) m/e: 971.3;measured m/e: 971.4.

Preparation of5-O-(5-Azido-5,6-dideoxy-2,3-O-dibenzoyi-α-L-talofuranosyl)-3,4,6,6-tetra-O-acetyl-2,3-diazido-1-N—[(S)-4-azido-2-O-acetyl-butanoyilparomamine(Compound (S)-23)

Anhydrous CH₂Cl₂ (15 ml) was added to a powdered, flame-dried 4 Åmolecular sieves (2.0 grams), followed by the addition acceptor Compound20 (1.0 grams, 0.0014 mol) and donor Compound (S)-17 (2.2 grams, 0.0042mol). The reaction mixture was stirred for 10 minutes at roomtemperature and was then cooled to −20° C. A catalytic amount ofBF3-Et₂O (0.1 ml) was added and the mixture was stirred at −15° C. andthe reaction progress was monitored by TLC, which indicated thecompletion after 60 minutes. The reaction mixture was diluted with ethylacetate and washed with saturated NaHCO₃ and brine. The combined organiclayer was dried over MgSO₄, evaporated and subjected to columnchromatography (EtOAc/Hexane) to obtain Compound (S)-23 (1.19 grams) in79% yield.

¹HNMR (500 MHz, CDCl₃): “Ring I” δ_(H) 3.63 (dd, 1H, J₁=4.2, J₂=10.4 Hz,H−2′), 4.18 (d, 1H, J=10.8 Hz, H−6′), 4.29 (dd, 1H, J₁=2.9, J₂=12.4 Hz,H−6′), 4.54 (m, 1H, H−5′), 5.09 (t, 1H, J=10.2 Hz, H−4′), 5.42 (t, 1H,J=10.2 Hz, H−3′), 5.84 (d, 1H, J=3.9 Hz, H−1′); “Ring II” δ_(H) 1.50(ddd, 1H, J₁=J₂=J₃=12.5 Hz, H−2_(ax)), 2.53 (td, 1H, J₁=4.5, J₂=12.5 Hz,H−2_(eq)), 3.60 (m, 1H, H−3), 3.74 (t, 1H, J=9.5 Hz, H−4), 3.96 (t, 1H,J=10.0 Hz, H−5), 4.06 (m, 1H, H−1), 4.93 (d, 1H, J=9.9 Hz, H−6); “RingIII” δ_(H) 1.33 (d, 3H, J=6.9 Hz, CH₃), 3.70 (m, 1H, H−5″), 4.33 (t, 1H,J=6.0 Hz, H−4″), 5.55 (dd, 1H, J₁=4.9, J₂=7.7 Hz, H−3″), 5.57 (m, 2H,H−2″ and H−1″). Additional peaks in the spectrum were identified asfollows: δ_(H) 2.04-2.10 (m, 2H, H−8 and H−8), 2.06 (s, 3H, OAc), 2.09(s, 6H, OAc), 2.26 (s, 3H, OAc), 2.35 (s, 3H, OAc), 3.37 (dd, 2H,J₁=6.0, J₂=7.5 Hz, H−9 and H−9), 5.20 (dd, 1H, J₁=1.5, J₂=8.5 Hz, H−7),6.69 (d, 1H, J=7.5 Hz, NH), 7.35 (t, 2H, J=8.0 Hz, Ar), 7.43 (t, 2H,J=8.0 Hz, Ar), 7.53 (t, 1H, J=8.0 Hz, Ar), 7.55 (t, 1H, J=8.0 Hz, Ar),7.87 (dd, 2H, J₁=1.1, J₂=8.2 Hz, Ar), 7.95 (dd, 2H, J₁=1.2, J₂=8.2 Hz,Ar).

¹³CNMR (125 MHz, CDCl₃): δc 15.4 (C-6″), 20.6 (4C, OAc), 20.9 (OAc),31.9 (C-1), 47.0, 48.5, 58.4, 58.7, 61.7, 61.8, 68.0, 68.2, 70.8, 70.9,71.4, 73.1, 74.7, 78.3, 79.7, 83.7, 96.7 (C-1′), 107.5 (C-1″), 128.4(Ar), 128.5 (2C, Ar), 128.7 (Ar), 129.6 (Ar), 129.7 (Ar), 133.5 (Ar),133.6 (Ar), 165.0 (C═O), 165.2 (C═O), 168.8 (C═O), 169.7 (2C, C═O),169.8 (C═O), 170.6 (C═O), 172.5 (C═O).

MALDI TOFMS calculated for C₄₆H₅₄N₁₃O₁₉ ([M+H]⁺) m/e: 1092.3; measuredm/e: 1092.3.

Preparation of5-O-(5-Azido-5,6-dideoxy-2,3-O-dibenzoyl-β-D-allofuranosyl)-3,4,6,6-tetra-O-acetyl-2,3-diazido-1-N-1(S)-4-azido-2-O-acetyl-butanoyl]paromamine(Compound (R)-24)

Anhydrous CH₂Cl₂ (15 ml) was added to a powdered, flame-dried 4 Åmolecular sieves (2.0 grams), followed by the addition acceptor Compound20 (1.0 grams, 0.0014 mol) and donor Compound (R)-18 (2.2 grams, 0.0042mol). The reaction mixture was stirred for 10 minutes at roomtemperature and was then cooled to −20° C. A catalytic amount ofBF3-Et₂O (0.1 ml) was added and the mixture was stirred at −15° C. andthe reaction progress was monitored by TLC, which indicated thecompletion after 60 minutes. The reaction mixture was diluted with ethylacetate and washed with saturated NaHCO₃ and brine. The combined organiclayer was dried over MgSO₄, evaporated and subjected to columnchromatography (EtOAc/Hexane) to obtain Compound (R)-24 (1.27 grams) in89% yield.

¹HNMR (500 MHz, CDCl₃): “Ring I” δ_(H) 3.53 (dd, 1H, J₁=4.7, J₂=10.7 Hz,H−2′), 4.18 (d, 1H, J=10.1 Hz, H−6′), 4.30 (dd, 1H, J₁=3.9, J₂=12.3 Hz,H−6′), 4.56 (m, 1H, H−5′), 5.09 (t, 1H, J=10.2 Hz, H−4′), 5.44 (t, 1H,J=9.7 Hz, H−3′), 5.84 (d, 1H, J=3.9 Hz, H−1′); “Ring II” δ_(H) 1.48(ddd, 1H, J₁=J₂=J₃=12.5 Hz, H−2_(ax)), 2.52 (td, 1H, J₁=4.5, J₂=12.5 Hz,H−2_(eq)), 3.60 (m, 1H, H−3), 3.74 (t, 1H, J=9.5 Hz, H−4), 4.00-4.08 (m,2H, H−5 and H−1), 4.93 (t, 1H, J=9.9 Hz, H−6); “Ring III”δ_(H) 1.41 (d,3H, J=6.9 Hz, CH₃), 3.83 (m, 1H, H−5″), 4.37 (dd, 1H, J₁=4.1, J₂=5.7 Hz,H−4″), 5.60 (t, 1H, J=6.5 Hz, H−3″), 5.64 (d, 1H, J=6.5 Hz, H−2″), 5.70(s, 1H, H−1″). Additional peaks in the spectrum were identified asfollows: δ_(H) 2.04-2.10 (m, 2H, H−8 and H−8), 2.06 (s, 3H, OAc), 2.10(s, 3H, OAc), 2.11 (s, 3H, OAc), 2.22 (s, 3H, OAc), 2.27 (s, 3H, OAc),3.37 (dd, 2H, J₁=6.0, J₂=7.5 Hz, H−9 and H−9), 5.19 (dd, 1H, J₁=1.5,J₂=8.5 Hz, H−7), 6.69 (d, 1H, J=7.5 Hz, NH), 7.35-7.43 (m, 4H, Ar),7.53-7.59 (m, 2H, Ar), 7.87-7.92 (m, 4H, Ar).

¹³CNMR (125 MHz, CDCl₃): δc 15.3 (C-6″), 20.5 (OAc), 20.5 (OAc), 20.6(OAc), 20.7 (OAc), 20.8 (OAc), 30.4, 32.1, 47.0, 48.4, 58.2, 58.5, 61.6,61.7, 68.0, 68.1, 70.7, 70.8, 70.9, 73.4, 74.7, 78.0, 79.5, 83.3, 96.8(C-1′), 106.9 (C-1″), 128.4 (2C, Ar), 128.7 (2C, Ar), 129.5 (Ar), 129.6(Ar), 133.5 (2C, Ar), 164.9 (C═O), 165.2 (C═O), 168.9 (C═O), 169.6(C═O), 169.7 (C═O), 169.8 (C═O), 170.6 (C═O), 172.3 (C═O).

MALDI TOFMS calculated for C₄₆H₅₄N₁₃O₁₉ ([M+H]⁺) mle: 1092.3; measuredm/e: 1092.3.

Preparation of 5-O-(5-Amino-5,6-dideoxy-α-L-talofuranosyl)-paromamine(NB118)

The glycosylation product Compound (S)-21 (1.0 grams, 0.001 mol) wastreated with a solution of MeNH₂ (33% solution in EtOH, 50 ml and thereaction progress was monitored by TLC (EtOAc/MeOH 85:15), whichindicated completion after 8 hours. The reaction mixture was evaporatedto dryness and the residue was dissolved in a mixture of THF (5 ml) andaqueous NaOH (1 mM, 5.0 ml). The mixture was stirred at room temperaturefor 10 minutes, after which PMe₃ (1 M solution in THF, 5.0 ml, 5.0 mmol)was added. The reaction progress was monitored by TLC[CH₂Cl₂/MeOH/H₂O/MeNH₂ (33% solution in EtOH) 10:15:6:15], whichindicated completion after 1 hour. The product was purified by columnchromatography on a short column of silica gel. The column was washedwith the following solvents: THF (800 ml), CH₂Cl₂ (800 ml), EtOH (200ml), and MeOH (400 ml).

The product was then eluted with a mixture of 20% MeNH₂ (33% solution inEtOH) in 80% MeOH. Fractions containing the product were combined andevaporated to dryness. The residue was re-dissolved in a small volume ofwater and evaporated again (2-3 repeats) to afford the free amine formof NB118.

The analytically pure product was obtained by passing the above productthrough a short column of Amberlite CG50 (NH₄ ⁺ form). The column wasfirst washed with a mixture of MeOH/H₂O (3:2), then the product waseluted with a mixture of MeOH/H₂O/NR₄OH (80:10:10) to afford NB118(0.405 grams, 82% yield).

For the storage and biological tests, compound was converted to itssulfate salt form: the free base was dissolved in water, the pH wasadjusted around 7.0 with H₂SO₄ (0.1 N) and lyophilized. [α]_(D) ²⁰=+38.4(c=0.2, MeOH).

¹HNMR (500 MHz, CD₃OD): “Ring I” δ_(H) 2.64 (dd, 1H, J₁=3.7, J₂=10.4 Hz,H−2′), 3.27 (t, 1H, J=9.7 Hz, H−4′) 3.52 (t, 1H, J=10.8 Hz, H−3′), 3.67(dd, 1H, J₁=6.0, J₂=11.8 Hz, H−6′), 3.79 (m, 1H, H−5′), 3.87 (dd, 1H,J₁=2.0, J₂=11.9 Hz, H−6′) 5.20 (d, 1H, J=3.4 Hz, H−1′); “Ring II” δ_(H)1.20 (ddd, 1H, J₁=J₂=J₃=12.5 Hz, H−2_(ax)), 1.97 (td, 1H, J₁=4.5,J₂=12.5 Hz, H−2_(eq)), 2.64 (m, 1H, H−1), 2.78 (m, 1H, H−3), 3.21 (t,1H, J=9.3 Hz, H−6), 3.38 (t, 1H, J=9.5 Hz, H−4), 3.50 (t, 1H, J=9.2,H−5); “Ring III” δ_(H) 1.18 (d, 3H, J=6.2 Hz, CH₃), 2.96 (m, 1H, H−5″),3.57 (t, 1H, J=6.9 Hz, H−4″), 4.02 (t, 1H, J=5.5 Hz, H−3″), 4.06 (dd,1H, J₁=2.9, J₂=5.4 Hz, H−2″), 5.25 (d, 1H, J=2.7 Hz, H−1″).

¹³CNMR (125 MHz, CD₃OD): δc 19.3 (C-6″), 37.5 (C-1), 50.6, 52.3, 52.6,57.8, 62.7 (C-6′), 72.1, 72.2, 75.3, 75.4, 76.2, 78.6, 84.6, 87.4, 88.6,102.0 (C-1′), 109.5 (C-1″).

MALDI TOFMS calculated for C₁₈H₃₇N₄O₁₀ ([M+H]⁺) m/e: 469.2; measuredm/e: 469.2.

Preparation of 5-O-(5-Amino-5,6-dideoxy-β-D-allofuranosyl)-paromamine(NB119)

The glycosylation product Compound (R)-22 (1.0 grams, 0.001 mol) wastreated with a solution of MeNH₂ (33% solution in EtOH, 50 ml) and thereaction progress was monitored by TLC (EtOAc/MeOH 85:15), whichindicated completion after 8 hours. The reaction mixture was evaporatedto dryness and the residue was dissolved in a mixture of THF (5 ml) andaqueous NaOH (1 mM, 5.0 ml). The mixture was stirred at room temperaturefor 10 minutes, after which PMe₃ (1 M solution in THF, 5.0 ml, 5.0 mmol)was added. The reaction progress was monitored by TLC[CH₂Cl₂/MeOH/H₂O/MeNH₂ (33% solution in EtOH) 10:15:6:15], whichindicated completion after 1 hour. The product was purified by columnchromatography on a short column of silica gel. The column was washedwith the following solvents: THF (800 ml), CH₂Cl₂ (800 ml), EtOH (200ml), and MeOH (400 ml). The product was then eluted with a mixture of20% MeNH₂ (33% solution in EtOH) in 80% MeOH. Fractions containing theproduct were combined and evaporated to dryness. The residue wasre-dissolved in a small volume of water and evaporated again (2-3repeats) to afford the free amine form of NB119, also referred to asNB119.

The analytically pure product was obtained by passing the above productthrough a short column of Amberlite CG50 (NH₄ ⁺ form). The column wasfirst washed with a mixture of MeOH/H₂O (3:2), then the product waseluted with a mixture of MeOH/H₂O/NH₄OH (80:10:10) to afford NB119(0.398 grams, 80% yield).

For the storage and biological tests, compound was converted to itssulfate salt form: the free base was dissolved in water, the pH wasadjusted around 7.0 with H₂SO₄ (0.1 N) and lyophilized. [α]D²⁰=+37.0(c=0.2, MeOH).

¹HNMR (500 MHz, CD₃OD): “Ring I” δ_(H) 2.64 (dd, 1H, J₁=3.4, J₂=10.2 Hz,H−2′), 3.27 (t, 1H, J=9.1 Hz, H−4′), 3.52 (t, 1H, J=8.9 Hz, H−3′), 3.68(t, 1H, J=6.1 Hz, H−6′), 3.79 (m, 1H, H−5′), 3.87 (dd, 1H, J₁=2.5,J₂=12.2 Hz, H−6′), 5.20 (d, 1H, J=3.6 Hz, H−1′); “Ring IF” δ_(H) 1.21(ddd, 1H, J₁=J₂=J₃=12.5 Hz, H−2_(ax)), 1.97 (td, 1H, J₁=4.5, J₂=12.5 Hz,H−2_(eq)), 2.64 (m, 1H, H−1), 2.78 (m, 1H, H−3), 3.18 (t, 1H, J=9.1 Hz,H−6), 3.37 (t, 1H, J=9.5 Hz, H−4), 3.46 (t, 1H, J=9.2 Hz, H−5); “RingIII” δ_(H) 1.16 (d, 3H, J=6.2 Hz, CH₃), 3.09 (m, 1H, H−5″), 3.70 (t, 1H,J=5.3 Hz, H−4″), 4.04 (dd, 1H, J₁=3.3, J₂=5.3 Hz, H−2″), 4.15 (t, 1H,J=5.5 Hz, H−3″), 5.21 (d, 1H, J=2.7 Hz, H−1″).

¹³CNMR (125 MHz, CD₃OD): δc 18.8 (C-6″), 37.6 (C-1), 49.4, 52.1, 52.6,57.8, 62.8 (C-6′), 70.8, 72.1, 75.2, 75.4, 76.1, 78.4, 84.7, 87.8, 88.2,102.0 (C-1′), 109.5 (C-1″).

MALDI TOFMS calculated for C₁₈H₃₇N₄O₁₀ ([M+H]⁺) m/e: 469.2; measuredm/e: 469.2

Preparation of5-O-(5-Amino-5,6-dideoxy-α-L-talofuranosyl)-1-N—[(S)-4-amino-2-hydroxy-butanoyl]paromamine(NB/22)

The glycosylation product Compound (S)-23 (1.1 grams, 0.001 mol) wastreated with a solution of MeNH₂ (33% solution in EtOH, 50 ml) and thereaction progress was monitored by TLC (EtOAc/MeOH 85:15), whichindicated completion after 8 hours. The reaction mixture was evaporatedto dryness and the residue was dissolved in a mixture of THF (5 ml) andaqueous NaOH (1 mM, 5.0 ml). The mixture was stirred at room temperaturefor 10 minutes, after which PMe₃ (1 M solution in THF, 5.0 ml, 5.0 mmol)was added. The reaction progress was monitored by TLC[CH₂Cl₂/MeOH/H₂O/MeNH₂ (33% solution in EtOH) 10:15:6:15], whichindicated completion after 1 hour. The product was purified by columnchromatography on a short column of silica gel. The column was washedwith the following solvents: THF (800 ml), CH₂Cl₂ (800 ml), EtOH (200ml), and MeOH (400 ml). The product was then eluted with a mixture of20% MeNH₂ (33% solution in EtOH) in 80% MeOH. Fractions containing theproduct were combined and evaporated to dryness. The residue wasre-dissolved in a small volume of water and evaporated again (2-3repeats) to afford the free amine form of NB122.

The analytically pure product was obtained by passing the above productthrough a short column of Amberlite CG50 (NH₄ ⁺ form). The column wasfirst washed with a mixture of MeOH/H₂O (3:2), then the product waseluted with a mixture of MeOH/H₂O/NH₄OH (80:10:10) to afford NB122(0.450 grams, 79% yield).

For the storage and biological tests, compound was converted to itssulfate salt form: the free base was dissolved in water, the pH wasadjusted around 7.0 with H₂SO₄ (0.1 N) and lyophilized. [α]_(D) ²⁰=+35.4(c=0.2, H₂O).

¹HNMR (500 MHz, CD₃₀D) “Ring I” δ_(H) 2.65 (dd, 1H, J₁=3.7 and J₂=10.3Hz, H−2′), 3.26 (t, 1H, J=8.9 Hz, H−4′), 3.54 (t, 1H, J=9.2 Hz, H−3′),3.68 (dd, 1H, J₁=5.9 and J₂=11.8 Hz, H−6′), 3.80 (m, 1H, H−5′), 3.87(dd, 1H, J₁=1.7 and J₂=11.7 Hz, H−6′), 5.21 (d, 1H, J=3.3 Hz, H−1′);“Ring II” δ_(H) 1.34 (ddd, 1H, J₁=J₂=J₃=12.5 Hz, H−2_(ax)), 1.99 (td,1H, J₁=4.5 and J₂=12.5 Hz, H−2_(eq)), 2.84 (m, 1H, H−3), 3.40 (t, 1H,J=9.0 Hz, H−4), 3.50-3.59 (m, 2H, H−5 and H−6), 3.81 (m, 1H, H−1); “RingIII” δ_(H) 1.17 (d, 3H, J=6.7 Hz, CH₃), 2.95 (m, 1H, H−5″), 3.57 (t, 1H,J=6.5 Hz, H−4″), 4.01 (t, 1H, J=5.7 Hz, H−3″), 4.08 (dd, 1H, J₁=2.7 andJ₂=5.4 Hz, H−2″), 5.26 (d, 1H, J=2.5 Hz, H−1″). Additional peaks in thespectrum were identified as follows: δ_(H) 1.82 (m, 1H, H−8), 1.94 (m,1H, H−8), 2.83 (t, 2H, J=6.4 Hz, H−9 and H−9), 4.14 (dd, 1H, J₁=4.1 andJ₂=7.6 Hz, H−7).

¹³CNMR (125 MHz, CD₃OD): δc 19.2 (C-6″), 35.9, 37.8, 38.9, 50.8, 50.9,52.4, 57.8, 62.8, 71.7, 72.1, 72.3, 75.3, 75.4, 75.6, 76.3, 84.7, 86.9,88.6, 101.9 (C-1′), 109.9 (C-1″), 177.1 (C═O).

MALDI TOFMS calculated for C₂₂H₄₄N₅O₁₂ ([M+H]⁺) mle: 570.3; measuredm/e: 570.27.

Preparation of5-O-(5-Amino-5,6-dideoxy-β-D-allofuranosyl)-1-N—[(S)-4-amino-2-hydroxy-butanoyl]paromamine(NB123)

The glycosylation product Compound (R)-24 (1.2 grams, 0.0011 mol) wastreated with a solution of MeNH₂ (33% solution in EtOH, 50 ml) and thereaction progress was monitored by TLC (EtOAc/MeOH 85:15), whichindicated completion after 8 hours. The reaction mixture was evaporatedto dryness and the residue was dissolved in a mixture of THF (5 ml) andaqueous NaOH (1 mM, 5.0 ml). The mixture was stirred at room temperaturefor 10 minutes, after which PMe₃ (1 M solution in THF, 5.0 ml, 5.0 mmol)was added. The reaction progress was monitored by TLC[CH₂Cl₂/MeOH/H₂O/MeNH₂ (33% solution in EtOH) 10:15:6:15], whichindicated completion after 1 hour. The product was purified by columnchromatography on a short column of silica gel. The column was washedwith the following solvents: THF (800 ml), CH₂Cl₂ (800 ml), EtOH (200ml), and MeOH (400 ml). The product was then eluted with a mixture of20% MeNH₂ (33% solution in EtOH) in 80% MeOH. Fractions containing theproduct were combined and evaporated to dryness. The residue wasre-dissolved in a small volume of water and evaporated again (2-3repeats) to afford the free amine form of NB123.

The analytically pure product was obtained by passing the above productthrough a short column of Amberlite CG50 (NH₄ ⁺ form). The column wasfirst washed with a mixture of MeOH/H₂O (3:2), then the product waseluted with a mixture of MeOH/H₂O/NH₄OH (80:10:10) to afford NB123(0.510 grams, 82% yield).

For the storage and biological tests, compound was converted to itssulfate salt form: the free base was dissolved in water, the pH wasadjusted around 7.0 with H₂SO₄ (0.1 N) and lyophilized. [α]_(D) ²⁰=+32.2(c=0.2, H₂O).

¹HNMR (500 MHz, CD₃OD) “Ring I” δ_(H) 2.65 (dd, 1H, J₁=3.4, J₂=10.0 Hz,H−2′), 3.27 (t, 1H, J=9.0 Hz, H−4′), 3.54 (t, 1H, J=9.1 Hz, H−3′), 3.66(dd, 1H, J₁=6.0, J₂=12.0 Hz, H−6′), 3.81 (m, 1H, H−5′), 3.88 (dd, 1H,J₁=2.0, J₂=12.0 Hz, H−6′), 5.21 (d, 1H, J=3.5 Hz, H−1′); “Ring II” δ_(H)1.33 (ddd, 1H, J₁=J₂=J₃=12.5 Hz, H−2_(ax)), 1.99 (td, 1H, J1=4.5,J₂=12.5 Hz, H−2_(eq)), 2.85 (m, 1H, H−3), 3.39 (t, 1H, J=9.0 Hz, H−4),3.49-3.57 (m, 2H, H−5 and H−6), 3.82 (m, 1H, H−1); “Ring III” δ_(H) 1.16(d, 3H, J=6.7 Hz, CH₃), 3.09 (m, 1H, H−5″), 3.70 (t, 1H, J=5.4 Hz,H−4″), 4.08 (dd, 1H, J₁=2.6, J₂=5.1 Hz, H−2″), 4.14 (t, 1H, J=5.7 Hz,H−3″), 5.22 (d, 1H, J=2.7 Hz, H−1″). Additional peaks in the spectrumwere identified as follows: δ_(H) 1.82 (m, 1H, H−8), 1.94 (m, 1H, H−8),2.84 (t, 2H, J=7.2 Hz, H−9 and H−9), 4.15 (dd, 1H, J₁=4.0, J₂=7.5 Hz,H−7).

¹³CNMR (125 MHz, CD₃OD): δ_(H) 18.8 (C-6″), 35.9, 37.6, 38.9, 49.6,40.8, 52.3, 57.8, 62.8, 71.0, 71.6, 72.1, 75.2, 75.3, 75.4, 76.2, 85.0,87.1, 87.9, 101.9 (C-1′), 110.0 (C-1″), 177.0 (C═O).

MALDI TOFMS calculated for C₂₂H₄₄N₅O₁₂ ([M+H]⁺) m/e: 570.3; measuredm/e: 570.27.

Preparation of6′-(R)-Methyl-5-O-(5-azido-5,6-dideoxy-2,3-O-dibenzoyl-α-L-talofuranosyl)-3,4,6,6-tetra-O-acetyl-2,1,3-triazidoparomamine (Compound (S)-221)

Anhydrous CH₂Cl₂ (15 ml) was added to a powdered, flame-dried 4 Åmolecular sieves (2.0 grams), followed by the addition acceptor Compound219 (0.9 grams, 0.0015 mol) and donor Compound (S)-17 (2.0 grams, 0.0037mol). The reaction mixture was stirred for 10 minutes at roomtemperature and was then cooled to −20° C. A catalytic amount ofBF3-Et₂O (0.1 ml) was added and the mixture was stirred at −15° C. andthe reaction progress was monitored by TLC, which indicated thecompletion after 120 minutes. The reaction mixture was diluted withethyl acetate and washed with saturated NaHCO₃ and brine. The combinedorganic layer was dried over MgSO₄, evaporated and subjected to columnchromatography (EtOAc/Hexane) to obtain Compound (S)-221 (1.1 grams) in75% yield.

¹HNMR (500 MHz, CDCl₃): “Ring I” δ_(H) 1.27 (d, 3H, J=6.0 Hz, CH₃), 3.58(dd, 1H, J₁=5.5, J₂=10.5 Hz, H−2′), 4.45 (d, 1H, J=10.7 Hz, H−5′),4.96-5.02 (m, 2H, H−4′ and H−6′), 5.42 (t, 1H, J=9.6 Hz, H−3′), 5.95 (d,1H, J=3.7 Hz, H−1′); “Ring II” δ_(H) 1.51 (ddd, 1H, J1=J₂=J₃=12.5 Hz,H−2_(ax)), 2.41 (td, 1H, J₁=4.5, J₂=12.5 Hz, H−2_(eq)), 3.55 (m, 2H, H−1and H−3), 3.76 (t, 1H, J=9.4 Hz, H−4), 3.88 (t, 1H, J=9.0 Hz, H−5), 5.03(t, 1H, J=9.1 Hz, H−6); “Ring III” δ_(H) 1.27 (d, 3H, J=5.6 Hz, CH₃),3.76 (m, 1H, H−5″), 4.35 (dd, 1H, J₁=6.9, J₂=10.9 Hz, H−4″), 5.45 (t,1H, J=5.5 Hz, H−3″), 5.62 (m, 2H, H−2″ and H−1″). Additional peaks inthe spectrum were identified as follows: δ_(H) 2.08 (s, 3H, OAc), 2.09(s, 6H, OAc), 2.38 (s, 3H, OAc), 7.37 (t, 2H, J=7.8 Hz, Ar), 7.41 (t,2H, J=7.8 Hz, Ar), 7.53-7.60 (m, 2H, Ar), 7.89 (d, 2H, J=8.0 Hz, Ar),7.93 (d, 2H, J=8.2 Hz, Ar).

¹³CNMR (125 MHz, CDCl₃): δc 13.3 (C-7′), 15.4 (C-6″), 20.6 (2C, OAc),20.9 (OAc), 21.1 (OAc), 32.1 (C-2), 58.4, 58.8, 59.5, 61.7, 68.5, 69.0,70.1, 70.8. 71.8, 73.6, 74.6, 77.3, 79.6, 84.4, 96.0 (C-1′), 107.6(C-1″), 128.4 (Ar), 128.5 (Ar), 128.6 (Ar), 128.7 (Ar), 129.6 (Ar),129.7 (Ar), 133.5 (Ar), 133.6 (Ar), 164.9 (C═O), 165.3 (C═O), 169.7(C═O), 169.9 (C═O), 170.1 (C═O), 170.2 (C═O).

MALDI TOFMS calculated for C₄₁H₄₆N₁₂O₁₆ Na ([M+Na]⁺) m/e: 985.3;measured m/e: 985.4.

Preparation of6′-(R)-Methyl-5-O-(5-azido-5,6-dideoxy-2,3-O-dibenzoyl-β-D-allofuranosyl)-3′,4′,6′,6-tetra-O-acetyl-2′,1,3-triazidoparomamine (Compound (R)-222)

Anhydrous CH₂Cl₂ (15 ml) was added to a powdered, flame-dried 4 Åmolecular sieves (2.0 grams), followed by the addition acceptor Compound219 (1.0 grams, 0.0017 mol) and donor Compound (R)-18 (2.2 grams, 0.004mol). The reaction mixture was stirred for 10 minutes at roomtemperature and was then cooled to −20° C. A catalytic amount ofBF3-Et₂O (0.1 ml) was added and the mixture was stirred at −15° C. andthe reaction progress was monitored by TLC, which indicated thecompletion after 120 minutes. The reaction mixture was diluted withethyl acetate and washed with saturated NaHCO₃ and brine. The combinedorganic layer was dried over MgSO₄, evaporated and subjected to columnchromatography (EtOAc/Hexane) to obtain Compound (R)-222 (1.2 grams) in75% yield.

¹HNMR (500 MHz, CDCl₃): “Ring I” δ_(H) 1.28 (d, 3H, J=6.7 Hz, CH₃), 3.46(dd, 1H, J₁=4.5, J₂=10.4 Hz, H−2′), 4.47 (d, 1H, J=10.7 Hz, H−5′),4.96-5.02 (m, 2H, H−4′ and H−6′), 5.44 (t, 1H, J=9.6 Hz, H−3′), 5.93 (d,1H, J=3.3 Hz, H−1′); “Ring II” δ_(H) 1.50 (ddd, 1H, J₁=J₂=J₃=12.5 Hz,H−2_(ax)), 2.41 (td, 1H, J₁=4.5 and J₂=12.5 Hz, H−2_(eq)), 3.56 (m, 2H,H−1 and H−3), 3.76 (t, 1H, J=10.0 Hz, H−4), 3.92 (t, 1H, J=9.5 Hz, H−5),5.04 (t, 1H, J=9.6 Hz, H−6); “Ring III” δ_(H) 1.42 (d, 3H, J=6.9 Hz,CH₃), 3.78 (m, 1H, H−5″), 4.40 (t, 1H, J=4.6 Hz, H−4″), 5.50 (t, 1H,J=5.0 Hz, H−3″), 5.59 (t, 1H, J=3.7 Hz, H−2″), 5.64 (s, 1H, H−1″).Additional peaks in the spectrum were identified as follows: δ_(H) 2.09(s, 9H, OAc), 2.33 (s, 3H, OAc), 7.37-7.41 (m, 4H, Ar), 7.56 (m, 2H,Ar), 7.92 (d, 4H, J=8.0 Hz Ar).

¹³CNMR (125 MHz, CDCl₃): δ_(H) 13.3 (C-7′), 15.0 (C-6″), 20.6 (OAc),20.7 (OAc), 20.8 (OAc), 21.2 (OAc), 32.1 (C-2), 58.1, 58.2, 58.8, 61.5,68.9, 70.2, 70.6, 71.4, 73.8, 74.6, 77.0, 77.1, 79.4, 83.9, 96.1 (C-1′),107.0 (C-1″), 128.4 (2C, Ar), 128.7 (2C, Ar), 129.6 (2C, Ar), 133.5(Ar), 133.6 (Ar), 164.9 (C═O), 165.4 (C═O), 169.8 (C═O), 169.9 (2C,C═O), 170.1 (C═O).

MALDI TOFMS calculated for C₄₁H₄₆N₁₂O₁₆Na ([M+Na]⁺) m/e: 985.3; measuredm/e: 985.4.

Preparation of6′-(R)-Methyl-5-O-(5-azido-5,6-dideoxy-2,3-O-dibenzoyl-α-L-talofuranosyl)-3,4,6,6-tetra-O-acetyl-2,3-diazido-1-N—[(S)-4-azido-2-O-acetyl-butanoyl]paromamine(Compound (S)-223)

Anhydrous CH₂Cl₂ (15 ml) was added to a powdered, flame-dried 4 Åmolecular sieves (2.0 grams), followed by the addition acceptor Compound220 (1.0 grams, 0.0014 mol) and donor Compound (S)-17 (2.5 grams, 0.0046mol). The reaction mixture was stirred for 10 minutes at roomtemperature and was then cooled to −20° C. A catalytic amount ofBF3-Et₂O (0.1 ml) was added and the mixture was stirred at −15° C. andthe reaction progress was monitored by TLC, which indicated thecompletion after 60 minutes. The reaction mixture was diluted with ethylacetate and washed with saturated NaHCO₃ and brine. The combined organiclayer was dried over MgSO₄, evaporated and subjected to columnchromatography (EtOAc/Hexane) to obtain Compound (S)-223 (1.1 grams) in73% yield.

¹HNMR (500 MHz, CDCl₃): ¹HNMR (500 MHz, CDCl₃): “Ring I” δ_(H) 1.27 (d,3H, J=5.2 Hz, CH₃), 3.54 (dd, 1H, J₁=4.3, J₂=10.5 Hz, H−2′), 4.45 (dd,1H, J₁=1.8, J₂=10.6 Hz, H−5′), 4.96-5.02 (m, 2H, H−4′ and H−6′), 5.43(t, 1H, J=9.4 Hz, H−3′), 5.94 (d, 1H, J=3.7 Hz, H−1′); “Ring II” δ_(H)1.44 (ddd, 1H, J₁=J₂=J₃=12.5 Hz, H−2_(ax)), 2.52 (td, 1H, J₁=4.5,J₂=12.5 Hz, H−2_(eq)), 3.60 (m, 1H, H−3), 3.66 (t, 1H, J=4.5 Hz, H−4),3.99 (t, 1H, J=6.4 Hz, H−5), 4.05 (m, 1H, H−1), 4.94 (t, 1H, J=9.2 Hz,H−6); “Ring III” δ_(H) 1.32 (d, 3H, J=6.9 Hz, CH₃), 3.72 (m, 1H, H−5″),4.32 (dd, 1H, J₁=5.85, J₂=8.0 Hz, H−4″), 5.55 (dd, 1H, J₁=4.7, J₂=7.4Hz, H−3″), 5.65 (m, 2H, H−2″ and H−1″). Additional peaks in the spectrumwere identified as follows: δ_(H) 2.04-2.10 (m, 2H, H−8 and H−8), 2.11(m, 9H, OAc), 2.22 (s, 3H, OAc), 2.30 (s, 3H, OAc), 3.37 (t, 2H, J=6.8Hz, H−9 and H−9), 5.20 (t, 1H, J=4.85 Hz, H−7), 6.70 (d, 1H, J=7.5 Hz,NH), 7.35 (t, 2H, J=7.6 Hz, Ar), 7.43 (t, 2H, J=7.8 Hz, Ar), 7.53-7.61(m, 2H, Ar), 7.86 (dd, 2H, J₁=1.1, J₂=8.2 Hz, Ar), 7.95 (dd, 2H, J₁=1.2,J₂=8.2 Hz, Ar).

¹³CNMR (125 MHz, CDCl₃): δc 13.5 (C-7′), 15.5 (C-6″), 20.6 (3C, OAc),20.9 (OAc), 21.1 (OAc), 30.4, 32.2 (C-1), 47.0, 48.4, 58.6, 58.7, 61.6,68.6, 69.0, 70.3, 70.8 (2C), 71.4, 73.1, 74.7, 77.5, 79.8, 83.6, 96.3(C-1′), 107.4 (C-1″), 128.4 (Ar), 128.5 (Ar), 128.7 (2C, Ar), 129.6(Ar), 129.7 (Ar), 133.5 (Ar), 133.6 (Ar), 165.0 (C═O), 165.2 (C═O),168.8 (C═O), 169.7 (2C, C═O), 169.9 (C═O), 170.0 (C═O), 172.4 (C═O).

MALDI TOFMS calculated for C₄₇H₅₅N₁₃O₁₉ Na ([M+Na]⁺) m/e: 1128.4;measured m/e: 1128.2.

Preparation of6′-(R)-Methyl-5-O-(5-azido-5,6-dideoxy-2,3-O-dibenzoyl-β-D-allofuranosyl)-3′,4′,6′,6-tetra-O-acetyl-2′,3-diazido-1-N—[(S)-4-azido-2-O-acetyl-butanoyl]paromamine(Compound (R)-224)

Anhydrous CH₂Cl₂ (15 ml) was added to a powdered, flame-dried 4 Åmolecular sieves (2.0 grams), followed by the addition acceptor Compound220 (1.0 grams, 0.0014 mol) and donor Compound (R)-18 (2.5 grams, 0.0046mol). The reaction mixture was stirred for 10 minutes at roomtemperature and was then cooled to −20° C. A catalytic amount ofBF₃-Et₂O (0.1 ml) was added and the mixture was stirred at −15° C. andthe reaction progress was monitored by TLC, which indicated thecompletion after 90 minutes. The reaction mixture was diluted with ethylacetate and washed with saturated NaHCO₃ and brine. The combined organiclayer was dried over MgSO₄, evaporated and subjected to columnchromatography (EtOAc/Hexane) to obtain Compound (R)-224 (1.15 grams) in76% yield.

¹HNMR (500 MHz, CDCl₃): ¹HNMR (500 MHz, CDCl₃): ¹HNMR (500 MHz, CDCl₃):“Ring I” δ_(H) 1.28 (d, 3H, J=6.6 Hz, CH₃), 3.43 (dd, 1H, J₁=4.3,J₂=10.6 Hz, H−2′), 4.49 (dd, 1H, J₁=2.2, J₂=10.7 Hz, H−5′), 4.96-5.02(m, 2H, H−4′ and H−6′), 5.45 (t, 1H, J=10.6 Hz, H−3′), 5.92 (d, 1H,J=3.7 Hz, H−1′); “Ring II” δ_(H) 1.42 (ddd, 1H, J₁=J₂=J₃=12.5 Hz,H−2_(ax)), 2.52 (td, 1H, J₁=4.5, J₂=12.5 Hz, H−2_(eq)), 3.64 (m, 1H,H−3), 3.76 (t, 1H, J=4.5 Hz, H−4), 4.05 (m, 2H, H−1 and H−5), 4.93 (t,1H, J=10.0 Hz, H−6); “Ring III” δ_(H) 1.39 (d, 3H, J=6.4 Hz, CH₃), 3.85(m, 1H, H−5″), 4.36 (dd, 1H, J₁=4.3, J₂=6.3 Hz, H−4″), 5.63 (m, 2H, H−2″and H−3″), 5.67 (s, 1H, H−1″). Additional peaks in the spectrum wereidentified as follows: δ_(H) 2.04-2.10 (m, 2H, H−8 and H−8), 2.08 (s,3H, OAc), 2.09 (s, 3H, OAc), 2.10 (s, 3H, OAc), 2.21 (s, 3H, OAc), 2.25(s, 3H, OAc), 3.37 (t, 2H, J=6.7 Hz, H−9 and H−9), 5.18 (t, 1H, J=5.0Hz, H−7), 6.66 (d, 1H, J=7.5 Hz, NH), 7.38-7.42 (m, 4H, Ar), 7.53-7.59(m, 2H, Ar), 7.89-7.92 (m, 4H, Ar).

¹³CNMR (125 MHz, CDCl₃): δc 13.5 (C-7′), 15.2 (C-6″), 20.6 (3C, OAc),20.8 (OAc), 21.1 (OAc), 30.4, 32.4 (C-1), 47.0, 48.4, 58.1, 58.7, 61.4,68.6, 69.0, 70.3, 70.5, 70.8, 70.9, 73.4, 74.8, 77.2, 79.6, 83.3, 96.3(C-1′), 106.9 (C-1″), 128.4 (2C, Ar), 128.7 (2C, Ar), 129.5 (Ar), 129.6(Ar), 133.5 (2C, Ar), 164.9 (C═O), 165.2 (C═O), 168.8 (C═O), 169.7 (2C,C═O), 169.9 (C═O), 170.0 (C═O), 172.3 (C═O).

MALDI TOFMS calculated for C₄₇H₅₅N₁₃O₁₉ Na ([M+Na]⁺) m/e: 1128.4;measured m/e: 1128.4.

Preparation of6′-(R)-Methyl-5-O-(5-amino-5,6-dideoxy-α-L-talofuranosyl)-paromamine(NB124)

The glycosylation product Compound (S)-221 (1.0 grams, 0.001 mol) wastreated with a solution of MeNH₂ (33% solution in EtOH, 50 ml) and thereaction progress was monitored by TLC (EtOAc/MeOH 85:15), whichindicated completion after 8 hours. The reaction mixture was evaporatedto dryness and the residue was dissolved in a mixture of THF (5 ml) andaqueous NaOH (1 mM, 5.0 ml). The mixture was stirred at room temperaturefor 10 minutes, after which PMe₃ (1 M solution in THF, 5.0 ml, 5.0 mmol)was added. The reaction progress was monitored by TLC[CH₂Cl₂/MeOH/H₂O/MeNH₂ (33% solution in EtOH) 10:15:6:15], whichindicated completion after 1 hour. The product was purified by columnchromatography on a short column of silica gel. The column was washedwith the following solvents: THF (800 ml), CH₂Cl₂ (800 ml), EtOH (200ml), and MeOH (400 ml). The product was then eluted with a mixture of20% MeNH₂ (33% solution in EtOH) in 80% MeOH. Fractions containing theproduct were combined and evaporated to dryness. The residue wasre-dissolved in a small volume of water and evaporated again (2-3repeats) to afford the free amine form of NB124.

Analytically pure product was obtained by passing the above productthrough a short column of Amberlite CG50 (NH₄ ⁺ form). The column wasfirst washed with a mixture of MeOH/H₂O (3:2), then the product waseluted with a mixture of MeOH/H₂O/NH₄OH (80:10:10) to afford NB124(0.400 grams, 79% yield).

For storage and biological tests, compound was converted to its sulfatesalt form: the free base was dissolved in water, the pH was adjustedaround 7.0 with H₂SO₄ (0.1 N) and lyophilized.

¹HNMR (500 MHz, CD₃OD): ¹HNMR (500 MHz, CD₃OD): “Ring I” δ_(H) 1.21 (d,3H, J=5.8 Hz, CH₃), 2.61 (dd, 1H, J₁=3.5, J₂=10.0 Hz, H−2′), 3.22 (t,1H, J=10.0 Hz, H−4′), 3.51 (t, 1H, J=8.9 Hz, H−3′), 3.81 (dd, 1H,J₁=3.0, J₂=10.0 Hz, H−5′), 4.12 (m, 1H, H−6′), 5.20 (d, 1H, J=3.3 Hz,H−1′); “Ring II” δ_(H) 1.18 (ddd, 1H, J₁=J₂=J₃=12.5 Hz, H−2_(ax)), 1.98(td, 1H, J₁=4.5, J₂=12.5 Hz, H−2_(eq)), 2.63 (m, 1H, H−1), 2.79 (m, 1H,H−3), 3.19 (t, 1H, J=9.7 Hz, H−6), 3.38 (t, 1H, J=9.3 Hz, H−4), 3.48 (t,1H, J=9.2 Hz, H−5); “Ring III” δ_(H) 1.18 (d, 3H, J=6.3 Hz, CH₃), 2.95(m, 1H, H−5″), 3.57 (t, 1H, J=6.4 Hz, H−4″), 4.03 (t, 1H, J=5.6 Hz,H−3″), 4.07 (m, 1H, H−2″), 5.25 (d, 1H, J=2.5 Hz, H−1″).

¹³CNMR (125 MHz, CD₃OD): δc 16.9 (C-7′), 19.3 (C-6″), 37.5 (C-1), 50.6,52.3, 52.6, 57.8, 67.8, 72.2, 73.6, 75.5, 76.2, 76.7, 78.6, 84.6, 87.3,88.6, 101.9 (C-1′), 109.6 (C-1″)

MALDI TOFMS calculated for C₁₉H₃₉N₄O₁₀ ([M+H]⁺) m/e: 483.3; measuredm/e: 483.2.

Preparation of6′-(R)-Methyl-5-O-(5-amino-5,6-dideoxy-β-D-allofuranosyl)-paromamine(NB125)

The glycosylation product Compound (R)-222 (1.0 grams, 0.001 mol) wastreated with a solution of MeNH₂ (33% solution in EtOH, 50 ml) and thereaction progress was monitored by TLC (EtOAc/MeOH 85:15), whichindicated completion after 8 hours. The reaction mixture was evaporatedto dryness and the residue was dissolved in a mixture of THF (5 ml) andaqueous NaOH (1 mM, 5.0 ml). The mixture was stirred at room temperaturefor 10 minutes, after which PMe₃ (1 M solution in THF, 5.0 ml, 5.0 mmol)was added. The reaction progress was monitored by TLC[CH₂Cl₂/MeOH/H₂O/MeNH₂ (33% solution in EtOH) 10:15:6:15], whichindicated completion after 1 hour. The product was purified by columnchromatography on a short column of silica gel. The column was washedwith the following solvents: THF (800 ml), CH₂Cl₂ (800 ml), EtOH (200ml), and MeOH (400 ml). The product was then eluted with a mixture of20% MeNH₂ (33% solution in EtOH) in 80% MeOH. Fractions containing theproduct were combined and evaporated to dryness. The residue wasre-dissolved in a small volume of water and evaporated again (2-3repeats) to afford the free amine form of NB125.

Analytically pure product was obtained by passing the above productthrough a short column of Amberlite CG50 (NH₄ ⁺ form). The column wasfirst washed with a mixture of MeOH/H₂O (3:2), then the product waseluted with a mixture of MeOH/H₂O/NR₄OH (80:10:10) to afford NB125(0.398 grams, 79% yield).

For storage and biological tests, compound was converted to its sulfatesalt form: the free base was dissolved in water, the pH was adjustedaround 7.0 with H₂SO₄ (0.1 N) and lyophilized.

¹HNMR (500 MHz, CD₃OD): “Ring I” δ_(H) 1.22 (d, 3H, J=5.8 Hz, CH₃), 2.61(dd, 1H, J₁=2.5, J₂=9.6 Hz, H−2′), 3.22 (t, 1H, J=9.8 Hz, H−4′), 3.50(t, 1H, J=9.9 Hz, H−3′), 3.83 (dd, 1H, J₁=3.0, J₂=10.1 Hz, H−5′), 4.12(m, 1H, H−6′), 5.20 (d, 1H, J=3.3 Hz, H−1′); “Ring II” δ_(H) 1.21 (ddd,1H, J₁=J₂=J₃=12.5 Hz, H−2_(ax)), 1.98 (td, 1H, J₁=4.5, J₂=12.5 Hz,H−2_(eq)), 2.65 (m, 1H, H−1), 2.78 (m, 1H, H−3), 3.18 (t, 1H, J=9.3 Hz,H−6), 3.38 (t, 1H, J=9.1 Hz, H−4), 3.46 (t, 1H, J=9.2 Hz, H−5); “RingIII” δ_(H) 1.17 (d, 3H, J=6.4 Hz, CH₃), 3.10 (m, 1H, H−5″), 3.71 (t, 1H,J=5.0 Hz, H−4″), 4.06 (t, 1H, J=5.6 Hz, H−2″), 4.16 (t, 1H, J=3.0 Hz,H−3″), 5.20 (d, 1H, J=3.0 Hz, H−1″).

¹³CNMR (125 MHz, CD₃OD): δc 16.6 (C-7′), 18.7 (C-6″), 37.6 (C-1), 49.5,52.2, 52.5, 57.8, 67.8, 70.8, 73.6, 75.4, 76.1, 76.7, 78.4, 84.7, 87.5,88.0, 101.9 (C-1′), 109.6 (C-1″).

MALDI TOFMS calculated for C₁₉H₃₉N₄O₁₀ ([M+H]⁺) mle: 483.3; measuredm/e: 483.2.

Preparation of6′-(R)-Methyl-5-O-(5-amino-5,6-dideoxy-α-L-talofuranosyl)-1-N—[(S)-4-amino-2-hydroxy-butanoyl]paromamine(NB127)

The glycosylation product Compound (S)-223 (1.05 grams, 0.001 mol) wastreated with a solution of MeNH₂ (33% solution in EtOH, 50 ml) and thereaction progress was monitored by TLC (EtOAc/MeOH 85:15), whichindicated completion after 8 hours. The reaction mixture was evaporatedto dryness and the residue was dissolved in a mixture of THF (5 ml) andaqueous NaOH (1 mM, 5.0 ml). The mixture was stirred at room temperaturefor 10 minutes, after which PMe₃ (1 M solution in THF, 5.0 ml, 5.0 mmol)was added. The reaction progress was monitored by TLC[CH₂Cl₂/MeOH/H₂O/MeNH₂(33% solution in EtOH) 10:15:6:15], whichindicated completion after 1 hour. The product was purified by columnchromatography on a short column of silica gel. The column was washedwith the following solvents: THF (800 ml), CH₂Cl₂ (800 ml), EtOH (200ml), and MeOH (400 ml). The product was then eluted with a mixture of20% MeNH₂ (33% solution in EtOH) in 80% MeOH. Fractions containing theproduct were combined and evaporated to dryness. The residue wasre-dissolved in a small volume of water and evaporated again (2-3repeats) to afford the free amine form of NB127.

Analytically pure product was obtained by passing the above productthrough a short column of Amberlite CG50 (NH₄ ⁺ form). The column wasfirst washed with a mixture of MeOH/H₂O (3:2), then the product waseluted with a mixture of MeOH/H₂O/NH₄OH (80:10:10) to afford NB127(0.480 grams, 86% yield).

For storage and biological tests, compound was converted to its sulfatesalt form: the free base was dissolved in water, the pH was adjustedaround 7.0 with H₂SO₄ (0.1 N) and lyophilized.

¹HNMR (500 MHz, CD₃OD) “Ring I” δ_(H) 1.21 (d, 3H, J=6.0 Hz, CH₃), 2.63(dd, 1H, J₁=3.5, J₂=10.0 Hz, H−2′), 3.23 (t, 1H, J=8.9 Hz, H−4′), 3.52(t, 1H, J=9.9 Hz, H−3′), 3.82 (dd, 1H, J₁=3.0, J₂=10.0 Hz, H−5′), 4.13(m, 1H, H−6′), 5.22 (d, 1H, J=3.3 Hz, H−1′); “Ring II” δ_(H) 1.34 (ddd,1H, J₁=J₂=J₃=12.5 Hz, H−2_(ax)), 1.99 (td, 1H, J₁=4.5 and J₂=12.5 Hz,H−2_(eq)), 2.85 (m, 1H, H−3), 3.40 (t, 1H, J=8.8 Hz, H−4), 3.50-3.59 (m,2H, H−5 and H−6), 3.83 (m, 1H, H−1); “Ring III” δ_(H) 1.17 (d, 3H, J=6.6Hz, CH₃), 2.94 (m, 1H, H−5″), 3.56 (t, 1H, J=7.1 Hz, H−4″), 4.01 (t, 1H,J=5.7 Hz, H−3″), 4.09 (dd, 1H, J₁=2.7 and J₂=5.4 Hz, H−2″), 5.26 (d, 1H,J=2.5 Hz, H−1″). Additional peaks in the spectrum were identified asfollows: OH 1.82 (m, 1H, H−8), 1.95 (m, 1H, H−8), 2.83 (t, 2H, J=5.7 Hz,H−9 and H−9), 4.13 (dd, 1H, J₁=4.2 and J₂=7.6 Hz, H−7).

¹³CNMR (125 MHz, CD₃OD): δc 16.6 (C-7′), 19.2 (C-6″), 35.9, 37.8, 39.0,50.8, 50.9, 52.3, 57.8, 67.8, 71.7, 72.4, 73.6, 75.5, 75.6, 76.3, 76.8,84.8, 86.7, 88.6, 101.9 (C-1′), 110.0 (C-1″), 177.1 (C═O).

MALDI TOFMS calculated for C₂₃H₄₅N₅O₁₂Na ([M+Na]⁺) m/e: 606.3; measuredm/e: 606.6.

Preparation of6′-(R)-Methyl-5-O-(5-amino-5,6-dideoxy-β-D-allofuranosyl)-1-N—[(S)-4-amino-2-hydroxy-butanoyl]paromamine(NB128)

The glycosylation product Compound (R)-224 (1.12 grams, 0.001 mol) wastreated with a solution of MeNH₂ (33% solution in EtOH, 50 ml) and thereaction progress was monitored by TLC (EtOAc/MeOH 85:15), whichindicated completion after 8 hours. The reaction mixture was evaporatedto dryness and the residue was dissolved in a mixture of THF (5 ml) andaqueous NaOH (1 mM, 5.0 ml). The mixture was stirred at room temperaturefor 10 minutes, after which PMe₃ (1 M solution in THF, 5.0 ml, 5.0 mmol)was added. The reaction progress was monitored by TLC[CH₂Cl₂/MeOH/H₂O/MeNH₂ (33% solution in EtOH) 10:15:6:15], whichindicated completion after 1 hour. The product was purified by columnchromatography on a short column of silica gel. The column was washedwith the following solvents: THF (800 ml), CH₂Cl₂ (800 ml), EtOH (200ml), and MeOH (400 ml). The product was then eluted with a mixture of20% MeNH₂ (33% solution in EtOH) in 80% MeOH. Fractions containing theproduct were combined and evaporated to dryness. The residue wasre-dissolved in a small volume of water and evaporated again (2-3repeats) to afford the free amine form NB128.

Analytically pure product was obtained by passing the above productthrough a short column of Amberlite CG50 (NH₄ ⁺ form). The column wasfirst washed with a mixture of MeOH/H₂O (3:2), then the product waseluted with a mixture of MeOH/H₂O/NR₄OH (80:10:10) to afford NB128(0.500 grams, 84% yield).

For storage and biological tests, compound was converted to its sulfatesalt form: the free base was dissolved in water, the pH was adjustedaround 7.0 with H₂SO₄ (0.1 N) and lyophilized.

¹HNMR (500 MHz, CD₃OD) “Ring I” δ_(H) 1.22 (d, 3H, J=6.3 Hz, CH₃), 2.63(dd, 1H, J₁=3.8, J₂=10.0 Hz, H−2′), 3.22 (t, 1H, J=9.8 Hz, H−4′), 3.52(dd, 1H, J₁=8.6, J₂=10.3 Hz, H−3′), 3.83 (dd, 1H, J₁=3.1, J₂=10.2 Hz,H−5′), 4.13 (m, 1H, H−6′), 5.23 (d, 1H, J=3.7 Hz, H−1′); “Ring II” δ_(H)1.34 (ddd, 1H, J₁=J₂=J₃=12.5 Hz, H−2_(ax)), 1.99 (td, 1H, J₁=4.5 andJ₂=12.5 Hz, H−2_(eq)), 2.85 (m, 1H, H−3), 3.39 (t, 1H, J=8.8 Hz, H−4),3.49-3.56 (m, 2H, H−5 and H−6), 3.82 (m, 1H, H−1); “Ring III” δ_(H) 1.16(d, 3H, J=6.7 Hz, CH₃), 3.08 (m, 1H, H−5″), 3.69 (t, 1H, J=5.5 Hz,H−4″), 4.07 (dd, 1H, J₁=2.1, J₂=5.2 Hz, H−2″), 4.14 (t, 1H, J=5.7 Hz,H−3″), 5.21 (d, 1H, J=3.7 Hz, H−1″). Additional peaks in the spectrumwere identified as follows: δ_(H) 1.82 (m, 1H, H−8), 1.95 (m, 1H, H−8),2.84 (t, 2H, J=7.2 Hz, H−9 and H−9), 4.13 (dd, 1H, J₁=3.9, J₂=7.5 Hz,H−7).

¹³CNMR (125 MHz, CD₃OD): δ_(H) 16.6 (C-7′), 18.8 (C-6″), 36.0, 37.7,38.9, 49.6, 50.8, 52.3, 57.8, 67.8, 71.0, 71.7, 73.6, 75.5 (2C), 76.2,76.7, 85.0, 86.9, 87.9, 101.9 (C-1′), 110.0 (C-1″), 177.1 (C═O).

MALDI TOFMS calculated for C₂₃H₄₅N₅O₁₂Na ([M+Na]⁺) m/e: 606.3; measuredm/e: 606.6.

Example 2 Stop Codon Readthrough

As presented hereinabove, the efficiency of aminoglycosides-inducedreadthrough is highly dependent on: (i) the identity of stop codon(UGA>UAG>UAA), (ii) the identity of the first nucleotide immediatelydownstream from the stop codon (C>U>A G) and (iii) the local sequencecontext around the stop codon. Therefore, in attempts to provide broadunderstanding on structure-activity relationship of the designedstructures, a variety of constructs containing different sequencecontexts around premature stop codons were used. These exemplarysequences were derived from the PCDH15, CFTR, IDUA and Dystrophin genesthat underlie USH1, CF, HS and DMD, respectively. The prevalent nonsensemutations of these diseases that were chosen were: R3X and R245X forUSH1, G542X and W1282X for CF, Q70X for HS and R3381X for DMD, aspresented hereinbelow.

Readthrough Assays:

DNA fragments derived from PCDH15, CFTR, Dystrophin and IDUA cDNAs,including the tested nonsense mutation or the corresponding wild type(wt) codon, and four to six upstream and downstream flanking codons werecreated by annealing following pairs of complementary oligonucleotides:

Usher Syndrome:

p.R3Xmut/wt (wild type sequence SEQ ID NO: 1 and SEQ ID NO: 2)5′-GATCCCAGAAGATGTTTCGACAGTTTTATCTCTGGACAGAGCT-3′, and5′-CTGTCAGAGATAAAACTGTCGAAACATCTTCTG-3′; (mutant sequence SEQ ID NO: 3and SEQ ID NO: 4) GATCCCAGAAGATGTTTTGACAGTTTTATCTCTGGACAGAGCT and5′-CTGTCAGAGATAAAACTGTCAAAACATCTTCTG-3′. p.R245Xmut/wt (WT sequence SEQID NO: 5 and 6) 5′-GATCCAAAATCTGAATGAGAGGCGAACCACCACCACCACCCTCGAGC T-3′and 5′-CGAGGGTGGTGGTGGTTGTTCGCCTCTCATTCAGATTTTG-3′; (mutant sequence SEQID NO: 7 and SEQ ID NO: 8)5′-GATCCAAAATCTGAATGAGAGGTGAACCACCACCACCACCCTCGA GCT and5′-CGAGGGTGGTGGTGGTTGTTCACCTCTCATTCAGATTTTG-3′.

Cystic Fibrosis:

p.G542Xmut/wt 5′-TCGACCAATATAGTTCTTGGAGAAGGTGGAATCGAGCT-3′ and5′-CGATTCCACCTTCTCGAAGAACTATATTGG-3′ (wild type sequence SEQ ID NO: 9and SEQ ID NO: 10); 5′-TCGACCAATATAGTTCTTTGAGAAGGTGGAATCGAGCT-3′5′-CGATTCCACCTTCTCAAAGAACTATATTGG-3′ (mutant sequence SEQ ID NO: 11 andSEQ ID NO: 12). p.W1282Xmut/wt5′-TCGACAACTTTGCAACAGTGGAGGAAAGCCTTTGAGCT-3′ and5-CAAAGGCTTTCCTCCACTGTTGCAAAGTTG-3′(WT sequence SEQ ID NOs: 13 and 14);5′-TCGACAACTTTGCAACAGTGAAGGAAAGCCTTTGAGCT-3′ and5-CAAAGGCTTTCCTTCACTGTTGCAAAGTTG-3′ (mutant sequence SEQ ID NO: 15 andSEQ ID NO: 16).

Duchene Muscular Dystrophy (DMD):

p.R3381Xmut/wt 5′-TCGACAAAAAACAAATTTTGCACCAAAAGGTATGAGCT-3′ and5′-CATACCTTTTGGTGCAAAATTTGTTTTTTG-3′ (wild type sequence SEQ ID NO: 17and SEQ ID NO: 18); 5′-TCGACAAAAAACAAATTTTGAACCAAAAGGTATGAGCT-3′ and5′-CATACCTTTTGGTTCAAAATTTGTTTTTTG-3′ (mutant sequence SEQ ID NO: 19 andSEQ ID NO: 20). Hurler Syndrome: p.Q70Xmut/wt5′-TCGACCCTCAGCTGGGACCAGCAGCTCAACCTCGAGCT-3′ and5′-CGAGGTTGAGCTGCTGGTCCCAGCTGAGG-3′ (wild type sequence SEQ ID NO: 21and SEQ ID NO: 22); 5′-TCGACCCTCAGCTGGGACTAGCAGCTCAACCTCGAGCT-3′ and5′-CGAGGTTGAGCTGCTAGTCCCAGCTGAGG-3′ (mutant sequence SEQ ID NO: 23 andSEQ ID NO: 24).

The fragments were inserted in frame into the polylinker of the p2Lucplasmid between either BamHl and Sacl (p.R3X and p.R245X), or Sall andSacl (all the rest) restriction sites.

For the in vitro readthrough assays, the obtained plasmids, withaddition of the tested aminoglycosides were transcribed and translatedusing the TNT Reticulocyte Lysate Quick CoupledTranscription/Translation System. Luciferase activity was determinedafter 90 minutes of incubation at 30° C., using the Dual LuciferaseReporter Assay System (Promega™).

For the ex vivo readthrough assays, the constructs harboring the R3X,R245X, Q70X and W1282X mutations were transfected to HEK-293 cells withLipofectamine 2000 (Invitrogen) and addition of the tested compounds wasperformed 6 hours post transfection. The cells were harvested following16 hours of incubation with the tested aminoglycosides. Stop codonreadthrough was calculated as previously described (see, Grentzmann, G.et al., RNA, 1998, 4, p. 479.).

Readthrough Results:

Initially, the influence of the chiral C5″-methyl group on readthroughpotential was evaluated on the pseudo-trisaccharides NB118 and NB119 byusing a dual luciferase reporter assay system as described hereinabove.Briefly, DNA fragments were cloned between BamHl and Sacl restrictionsites of the p2luc vector and the obtained constructs were transcribedand translated using TNT quick coupled transcription/translation system.The amount of the translated products was evaluated using the dualluciferase reporter assay system and used to calculate the suppressionlevel. The results, which represent averages of at least threeindependent experiments, are summarized in FIGS. 2A-2F.

FIGS. 2A-2F present the results of the stop codon readthrough assayshowing comparative graphs of in vitro stop codon suppression levelsinduced by NB30 (marked by empty circles), NB118 (marked by blacktriangles), NB119 (marked by empty triangles) and the control druggentamicin (marked by black rectangles) in a series of nonsense mutationcontext constructs representing various genetic diseases (inparenthesis), wherein results pertaining to the R3X (USH1) construct areshown in FIG. 2A, R245X (USH1) in FIG. 2B, G542X (CF) in FIG. 2C, W1282X(CF) in FIG. 2D, Q70X (HS) in FIG. 2E, and wherein results pertaining tothe R3381X (DMD) construct are shown in FIG. 2F.

As can be seen in FIGS. A-F 2, in all the mutations tested, installationof (S)-5″-methyl group, as in NB118), on NB30 dramatically increases itsin vitro readthrough activity, whereas that of the (R)-5″-methyl group,as in NB119), is comparatively small. In addition, in all mutationstested (except G542X, see FIG. 2C), the readthrough activity of NB118was significantly better than that of the clinical drug gentamicin.

The same potency enhancement, attributed to the addition of the(S)-5″-methyl group, was explored in the case of NB54. To evaluate theimpact of the stereochemistry at C5″-position, both C5″-diastereomerswere synthesized, namely NB122 and NB123. Comparative in vitrosuppression tests of the pseudo-trisaccharides NB54, NB122, NB123, andthe control drug gentamicin were performed under the same experimentalconditions as described hereinabove for compounds NB30, NB54 and NB118,and the observed data (averages of at least three independentexperiments) are presented in FIGS. 3A-3F.

FIGS. 3A-3F present the results of the stop codon readthrough assayshowing comparative graphs of in vitro stop codon suppression levelsinduced by NB54 (marked by black circles), NB122 (marked by blacktriangles), NB123 (marked by empty triangles) and gentamicin (marked byblack rectangles) in a series of nonsense mutation context constructsrepresenting various genetic diseases (in parenthesis), wherein resultspertaining to the R3X (USH1) construct are shown in FIG. 3A, R245X(USH1) in FIG. 3B, G542X (CF) in FIG. 3C, W1282X (CF) in FIG. 3D, Q70X(HS) in FIG. 3E, and wherein results pertaining to the R3381X (DMD)construct are shown in FIG. 3F.

As can be seen in FIGS. 3A-3F, the efficacy of readthrough issubstantially different between different constructs and compoundstested, with no obvious dependence of readthrough effectiveness on theintroduced type of modification on aminoglycoside. Nevertheless, in allmutations tested (except R3X and Q70X, FIG. 3A and FIG. 3E), NB122induced the highest level of readthrough, followed by NB123, NB54, andgentamicin. The UGA C tetracodon sequence (R3X) showed the besttranslational readthrough than UGA A and UGA G, with the UAG Ctetracodon least efficient, in agreement with earlier observations.

To further evaluate the readthrough potential of NB122 and NB123, theiractivity was assayed in cultured mammalian cells using four differentdual luciferase reporter plasmids harboring the PCDH15-R3X andPCDH/5-R245X nonsense mutation of USH1, the IDUA-Q70X nonsense mutationof HS, and the CFTR-W1282X nonsense mutation of CF. These reporterconstructs were the same as presented hereinabove for the in vitrostudy, and have distinct advantage to control for differences in mRNAlevels between normal and nonsense-containing sequences over those ofsingle reporter or direct protein analysis.

The constructs were transfected into a human embryonic kidney cell line(HEK-293) and incubated with varying concentrations of NB122, NB123,NB54 and the control drug gentamicin, and the results are presented inFIGS. 4A-D.

FIGS. 4A-4D present ex vivo suppression of the PCDH15-R3X (FIG. 4A),PCDH/5-R245X (FIG. 4B), IDUA-Q70X (FIG. 4C), and CFTR-W1282X (FIG. 4D)nonsense mutations, effected by NB54 (marked by black circles), NB122(marked by black triangle), NB123 (marked by empty triangles) and thecontrol drug gentamicin (marked by black rectangles).

As described hereinabove, the constructs of p2luc plasmid harboring theR3X, R245X, Q70X and W1282X mutations were transfected to HEK-293 cellsusing lipofectamine2000 and the tested compounds were added 6 hours posttransfection. Cells were harvested after 16 hours incubation andluciferase activity was determined using the dual luciferase reporterassay system (Promega™). Stop codon readthrough was calculated asdescribed previously, and the results are averages of at least threeindependent experiments.

As can be seen in FIGS. 4A-4D, in all the mutations tested, the observedefficacy of aminoglycoside-induced readthrough was in the orderNB122≥NB123>NB54> gentamicin. This trend for NB122 and NB123 was similarto that observed for the suppression of the same stop mutations in vitro(see, FIGS. 3A-F), even though the gap of potency difference betweenNB122 and NB123 was smaller than the one observed for the suppression ofthe same mutations in cell-free extracts.

The significantly higher readthrough potencies observed for both NB122and NB123, over that of NB54 in R3X and Q70X (see, FIG. 4A and FIG. 4C),was considerably different to those of the same mutations in vitro (FIG.3A and FIG. 3E). This data may point to a better cell permeability ofboth NB122 and NB123 over that of NB54, due to the presence of the5″-methyl group.

Several combinations of the aforementioned pharmacophore points into onemolecule including N₁-AHB with (R)-6′-methyl group gave the knowncompound NB84, and N1-AHB with (S)— and (R)-5″-methyl groups gave theexemplary compounds according to some embodiments of the presentinvention, NB122 and NB123. All these exemplary compounds have beenshown to exhibit significantly improved readthrough activity than theparent structures while the cytotoxicity of the resulting novelstructures did not change significantly. One of the objectives of thepresent study was to test additional combinations of the above elements.As such the combination of (R)-6′-methyl group with either (S)-5″-methylgroup or (R)-5″-methyl group into one molecule. For that end exemplarycompounds NB124 and NB125 have been prepared and tested. The combinationof the latter two chiral methyl groups with N1-AHB group gave twoexemplary compounds NB127 and NB128.

As in the previous series, the influence of two chiral methyl groups onreadthrough potential was evaluated in vitro on thepseudo-trisaccharides NB124 [(R)-6′, (S)-5″] and NB125 [(R)-6′, (R)-5″]by using a dual luciferase reporter assay system as describedhereinabove, and the results are presented in FIGS. 5A-B and FIGS. 6A-F.

FIGS. 5A-5D present comparative plots of results of in vitro prematurestop codon mutation suppression assays of the CFTR-G542X (FIG. 5A andFIG. 5C), CFTR-W1282X (FIG. 5B and FIG. 5D) effected by exemplarycompounds according to some embodiments of the present invention NB124(marked by black circles), NB125 (marked by empty circles), NB127(marked by black triangles), NB128 (marked by empty triangles), NB74(marked by empty rhombs) NB84 (marked by black rhombs), and the controldrugs gentamicin (marked by black rectangles) and G418 (marked by emptyrectangles).

FIGS. 6A-6F present the results of the stop codon readthrough assayshowing comparative graphs of in vitro stop codon readthrough levelsinduced by NB124 (marked by black circles), NB125 (marked by emptycircles), NB74 (marked by empty rhombs) and the control drug gentamicin(marked by black rectangles) in a series of nonsense mutation contextconstructs representing various genetic diseases (in parenthesis),wherein results pertaining to the R3X (USH1) construct are shown in FIG.6A, R245X (USH1) in FIG. 6B, G542X (CF) in FIG. 6C, W1282X (CF) in FIG.6D, Q70X (HS) in FIG. 6E, and wherein results pertaining to the R3381X(DMD) construct are shown in FIG. 6F.

As can be seen in FIGS. 5A-5B and FIGS. 6A-6F, the addition of the(S)-5″-methyl group on the structure of the known compound NB74 toafford NB124 increases its in vitro readthrough activity significantly,whereas that of the (R)-5″-methyl group (in compound NB125) is smallercomparatively. In addition, in all mutations tested the readthroughactivity of NB124 was improved significantly compared to that of theclinical drug gentamicin. Thus, the two methyl groups (R)-6′-methyl and(S)-5″-methyl in compound NB124 are operating additively orsynergistically to enhance readthrough activity in comparison to NB30,NB74 and NB118. The conversions of either NB30 to NB74 to NB124 (namelythe addition of first (R)-6′-methyl group on NB30 to yield NB74 and thenfurther addition of (S)-5″-methyl on NB74 to yield NB124), or NB30 toNB118 to NB124 (namely the addition of first (S)-5″-methyl group on NB30to yield NB118 and then further addition of (R)-6′-methyl group on NB118to yield NB124), are affecting additively to increase the observedactivity of the resulted structures in a step-wise manner.

Interestingly, similar additive effect was also observed when the abovetwo methyl groups in NB124 and NB125 were combined with the N1-AHB groupto yield the compounds NB127 and NB128, respectively, as presented inFIGS. 5C-5D and FIGS. 7A-7F.

FIGS. 7A-7F present the results of the stop codon readthrough assayshowing comparative graphs of in vitro stop codon suppression levelsinduced by NB84 (marked by black rhombs), NB127 (marked by blacktriangles), NB128 (marked by empty triangles), G418 (marked by emptyrectangles) and gentamicin (marked by black rectangles) in a series ofnonsense mutation context constructs representing various geneticdiseases (in parenthesis), wherein results pertaining to the R3X (USH1)construct are shown in FIG. 7A, R245X (USH1) in FIG. 7B, G542X (CF) inFIG. 7C, W1282X (CF) in FIG. 7D, Q70X (HS) in FIG. 7E, and whereinresults pertaining to the R3381X (DMD) construct are shown in FIG. 7F.

As can be seen in FIGS. 5C-5D and FIGS. 7A-7F, NB127 which contains(S)-5″-methyl group is significantly potent than the NB128 containing(R)-5″-methyl group. In addition, both NB127 and NB128 are significantlystronger readthrough inducers than the corresponding counterparts notpossessing an AHB moiety in the N1 position (namely NB124 and NB125) andthe compound NB84 that contains only (R)-6′-mathyl and N1-AHB.

It is noted herein that in several mutations contests tested, such asG542X, W1282X and Q70X, NB127 exhibited similar or greater activity thanthat of G418, and further that in all the in vitro tests performed todate, G418 is considered the strongest readthrough inducer. Theobservation that NB127 can surpasses G416 activity, while exhibiting farlower cell toxicity than that of G418 (see the Table 2) demonstrates thebenefits conferred by compounds according to some embodiments of thepresent invention.

The observed in vitro activity data is further supported by ex vivocomparative activity tests shown in FIGS. 8-10.

FIGS. 8A-8D present comparative plots of results of ex vivo prematurestop codon mutation suppression assays conducted for the constructCFTR-G542X (FIGS. 8A and 8C), CFTR-W1282X (FIGS. 8B and 8D) effected byNB124 (marked by black circles), NB125 (marked by empty circles), NB127(marked by black triangles), NB128 (marked by empty triangles), NB74(marked by empty rhombs) NB84 (marked by black rhombs) and the controldrugs gentamicin (marked by black rectangles) and G418 (marked by emptyrectangles).

FIGS. 9A-9E present the results of the stop codon readthrough assayshowing comparative graphs of ex vivo stop codon suppression levelsinduced by NB124 (marked by black circles), NB125 (marked by emptycircles), NB74 (marked by black rhombs) and the control drugs gentamicin(marked by black rectangles) and G418 (marked by empty rectangles) in aseries of nonsense mutation context constructs representing variousgenetic diseases (in parenthesis), wherein results pertaining to the R3X(USH1) construct are shown in FIG. 9A, R245X (USH1) in FIG. 9B, Q70X(HS) in FIG. 9C, W1282X (CF) in FIG. 9D and G542X (CF) in FIG. 9E.

FIGS. 10A-10E present the results of the stop codon readthrough assayshowing comparative graphs of ex vivo stop codon suppression levelsinduced by NB127 (marked by black rectangles), NB128 (marked by emptytriangles), NB84 (marked by black rhombs) and the control drugsgentamicin (marked by black rectangles) and G418 (marked by emptyrectangles) in a series of nonsense mutation context constructsrepresenting various genetic diseases (in parenthesis), wherein resultspertaining to the R3X (USH1) construct are shown in FIG. 10A, R245X(USH1) in FIG. 10B, Q70X (HS) in FIG. 10C, W1282X (CF) in FIG. 10D andG542X (CF) in FIG. 10E.

As can be seen in FIGS. 8-10, in all the mutations tested, the observedefficacy of aminoglycoside-induced readthrough was in the order ofNB124>NB125>NB74> gentamicin and NB127 NB128>NB84>gentamicin. Thesetrends are similar to those observed for the suppression of the samestop mutations in vitro (see, FIGS. 5-7), even though the gap of potencydifference between the NB127 and NB128 was smaller than the one observedfor the suppression of the same mutations in vitro in cell-freeextracts.

Example 3 Cell Toxicity vs. Readthrough

In order to ensure suitable cell viability for each of the testedcompounds at the concentrations tested, cell toxicity was evaluated foreach compound by measuring the half-maximal-lethal concentration value(LC₅₀ values) in HEK-293 and HFF (human foreskin fibroblasts) cells.

The percentages of cell viability were calculated as the ratio betweenthe numbers of living cells in cultures grown in the presence of thetested compounds, versus cultures grown under the identical protocol butwithout the tested compound. The results represent averages of at leastthree independent experiments.

FIGS. 11A-11D present semi-logarithmic plots of in vitro translationinhibition in prokaryotic (marked by black circles) and eukaryotic(marked by empty circles) systems measured for NB118 (FIG. 11A), NB119(FIG. 11B) NB122 (FIG. 11C) and NB123 (FIG. 11D).

FIGS. 12A-12D present semi-logarithmic plots of the percentages of exvivo cell viability versus concentration of the tested compound inHEK-293 (FIG. 12A and FIG. 12C) and in human foreskin fibroblasts (HFF)(FIG. 12B and FIG. 12D) cells, for gentamicin (marked by emptyrectangles), NB118 (marked by empty circles), NB119 (marked by blackcircles), NB122 (marked by empty triangles), and NB123 (marked by blacktriangle).

The half-maximal lethal concentration (LC₅₀) values were obtained fromfitting concentration-response curves to the data of at least threeindependent experiments, using GraFit 5 software.

Prokaryotic and eukaryotic translation inhibition was quantified incoupled transcription/translation assays by using active luciferasedetection, performed as described hereinabove. The MIC values weredetermined by using the double-microdilution method, with two differentstarting concentrations of each tested compound (384 μg/mL and 6,144μg/mL). All the experiments were performed in duplicates and analogousresults were obtained in three different experiments. In all biologicaltests, all tested aminoglycosides were in their sulfate salt forms. Theconcentrations reported refer to that of the free amine form of eachaminoglycoside.

Table 1 presents comparative cell toxicity, eukaryotic and prokaryotictranslation inhibition, and antibacterial activity assays obtain forgentamicin, paromomycin, the previously reported NB30 and NB54, and theexemplary compounds NB118, NB119, NB122 and NB123.

TABLE 1 Antibacterial activity Translation inhibition MIC (μM)Prokaryotic Eukaryotic Cell toxicity B. subtilis E. coli system SystemLC₅₀ (mM) ATCC6633 R477/100 IC₅₀ (nM) IC₅₀ (μM) HFF HEK-293Aminoglycoside <0.75 6 28 ± 4 62 ± 9  3.2 ± 0.3  2.5 ± 0.3 Gentamicin1.2 22 51 ± 5 57 ± 4  3.1 ± 0.4  4.1 ± 0.5 Paromomycin 100 790 460 ± 5031 ± 4 21.8 ± 0.9 21.4 ± 3.9 NB30 70 588 160 ± 20 24 ± 1  7.8 ± 0.4  6.1± 0.6 NB54 83 2659 1960 ± 206  16 ± 1.3 21.8 ± 0.5 23.5 ± 0.6 NB118 784989 2132 ± 478  28 ± 1.1 20.1 ± 0.6 19.8 ± 0.4 NB119 33 1067 2266 ± 196 5.2 ± 0.7  8.1 ± 1.4 10.1 ± 0.8 NB122 33 1057 811 ± 59  4.6 ± 0.6 19.3± 1.5 13.9 ± 1.3 NB123

Comparison of the observed cell toxicity data in Table 1 with thereadthrough activity data in FIGS. 2-4, demonstrates that theinstallation of (S)-5″-methyl group either on NB30 to give NB118, or onNB54 to give NB122, does not significantly affect the cytotoxicity (LC₅₀values of 21.4 and 23.5 mM for NB30 and NB118 respectively, and 6.1 and10.1 mM for NB54 and NB122 respectively, respectively in HEK-293), whileit greatly increases the observed stop codon suppression activity(NB30<NB118 and NB54<NB122). The similar cell toxicity observed in thecase of NB122 and NB54 in HEK-293 and HFF cells (see, Table 1), togetherwith substantially elevated suppression activity of NB122 over that ofNB54 both in vitro and ex vivo in cultured cells, indicate that NB122may represent a more superior choice than NB54 in suppression therapy.

That the comparative ex-vivo suppression data in FIG. 4 shows only asmall preference of NB122 over that of NB123, while the cell toxicitydata in Table 1 indicate small (HEK-293 cells) to significantly (HFFcells) better cell toxicity profile of NB123 over that of NB122.Therefore, one may argue that in vivo performance of NB123 diastereomermight be even better than that of NB122. In addition, very recent studyon gentamicin demonstrated that the inversion of an absoluteconfiguration at a single carbon atom, from (S)-6′-gentamicin C₂ to(R)-6′-gentamicin C₂, significantly reduces cell toxicity and apparentnephrotoxicity of the (R)-diastereomer in comparison to that of(S)-diastereomer, as determined in cell culture and animal studies,while the bactericidal efficacy is not affected.

Based on these observations it is clear that additional toxicity tests,including nephrotoxicity and ototoxicity, the major drawbacks of knownaminoglycosides, can resolve this issue satisfactorily and validate theobserved benefit of either NB122 or NB123, over that of NB54 and overthat of gentamicin.

The impact of (S)-5″-methyl group on the elevated readthrough activitiesof NB118 and NB122 is further supported by the observed eukaryotictranslation inhibition data (see, Table 1). The efficacy with whichNB122 (half-maximal inhibitory concentration value IC₅₀=5.2 μM) inhibitseukaryotic translation is greater than that of NB118 (IC₅₀=16.0 μM) andNB54 (IC₅₀=24.0 μM), a similar trend to that observed for readthroughactivity, namely NB122>NB118>NB54 (see, FIGS. 2-4). In addition, thecomparison of IC₅₀ values of NB118 and NB122 to those of their parentstructures NB30 and NB54 (IC₅₀ values of 31 and 24 μM, respectively),reveals that NB118 and NB122 are 1.9-fold and 4.6-fold more specific tothe eukaryotic ribosome than their parents NB30 and NB54, indicatingthat the observed impact of (S)-5″-methyl group on the elevatedreadthrough activities of NB118 and NB122 is associated with theirincreased specificity to the eukaryotic ribosome.

Table 2 presents comparative results of cell toxicity, eukaryotic andprokaryotic translation inhibition, and antibacterial activity assaysobtain for gentamicin, G418, the previously reported NB74 and NB84, andthe exemplary compounds NB124, NB125, NB127 and NB128.

TABLE 2 Antibacterial activity Translation Inhibition MIC (μM)Prokaryotic Eukaryotic Cell toxicity B. subtilis E. coli system SystemLC₅₀ (mM) ATCC6633 R477/100 IC₅₀ (μM) IC₅₀ (μM) HFF HEK-293Aminoglycoside <0.75 6 0.028 ± 0.004 62 ± 9  3.21 ± 0.31 2.65 ± 0.54Gentamicin <1.25 9 0.009 ± 0.002  2 ± 0.3 1.59 ± 0.14 1.31 ± 0.06 G41842 680 1.130 ± 0.120  17 ± 0.6 21.34 ± 1.72  22.17 ± 1.06  NB74 70 5560.980 ± 0.070 2.8 ± 0.3 16.33 ± 0.47  5.77 ± 0.68 NB84 96 768 1.102 ±0.185 1.49 ± 0.08 4.75 ± 0.33 5.40 ± 0.45 NB124 96 1536 1.862 ± 0.1737.96 ± 0.27 7.59 ± 0.18 16.54 ± 3.10  NB125 192 384 1.753 ± 0.274 0.73 ±0.07 6.48 ± 0.26 5.09 ± 0.27 NB127 96 384 1.752 ± 0.145 0.89 ± 0.07 2.78± 0.11 5.35 ± 0.31 NB128

As can be seen in Table 2, comparison of the observed cell toxicity datain Table 2 with the readthrough activity data presented in FIGS. 8-10,demonstrates that compounds according to some embodiments of the presentinvention, such as NB124, NB125, NB127 and NB128 exhibit approximatelythe same level of cell toxicity in comparison to previously disclosedcompounds, with the exception of NB128 cytotoxicity in human foreskinfibroblast (HFF).

In addition, similar to previously disclosed compounds, the novel NB124,NB125, NB127 and NB128 compounds do not exhibit significantantibacterial activity both in E. coli and B. subtilis (see, Table 2above). These data are further supported by their drastically reducedinhibition of prokaryotic protein synthesis (Table 2) in comparison tostandard aminoglycoside antibiotics and thus are in accordance to ageneral trend that aminoglycosides with reduced inhibition ofprokaryotic translation are also less cytotoxic probably due to reducedinhibition of mitochondrial protein synthesis.

Example 4 Antibacterial Activity

Results of antimicrobial activity assays obtained for some exemplarycompounds according to embodiments of the present invention arepresented in Tables 1 and 2 hereinabove.

It has been shown previously that compounds such as NB30, NB54, NB74 andNB84 are about 10-fold weaker inhibitors of prokaryotic translation thangentamicin and paromomycin, and further exhibit almost no bactericidalactivity against both Gram-negative and Gram-positive bacteria. Thepresent experiments determine whether compounds according to someembodiments of the present invention, such as NB118, NB119, NB122,NB123, NB124, NB125, NB127 and NB128, retain similar properties.

Hence, exemplary compounds NB118, NB119, NB122, NB123, NB124, NB125,NB127 and NB128 were investigated as antibacterial agents against bothGram-negative (Escherichia coli) and Gram-positive (Bacillus subtilis)bacteria, together with their prokaryotic anti-translational activities(see, Table 1 and Table 2).

As can be seen in Table 1 and Table 2, the measured IC₅₀ values showthat the efficacy with which exemplary compounds according to someembodiments of the present invention, inhibit the prokaryotic ribosomeis significantly lower than that of paromomycin and gentamicin, inaccordance with the observed antibacterial data of this set ofcompounds; while gentamicin and paromomycin exhibit significantantibacterial activities against both E. coli and B. subtilis, exemplarycompounds according to some embodiments of the present invention lackconsiderable antibacterial activity.

The observed data with NB118, NB119, NB122, NB123, NB124, NB125, NB127and NB128 is similar to that observed for NB30, NB54, NB74 and NB84 andfurther support the previously reported correlation in aminoglycosidesbetween prokaryotic anti-translational activity and MIC values, namely,decreased inhibition of prokaryotic translation is associated with thedecrease in antibacterial activity.

Furthermore, the observed continued inability of NB30, NB54, NB74 andNB84, as well as of NB118, NB119, NB122, NB123, NB124, NB125, NB127 andNB128, to show significant antibacterial activity in conjunction withtheir decreased prokaryotic ribosome specificity, suggest that byreducing the specificity to prokaryotic ribosome, and thereby takingaway their antibacterial activity, their action on eukaryoticmitochondrial protein synthesis machinery may be reduced, and therebysignificantly reduce their toxic effects on humans. This view issupported by the fact that the mammalian mitochondrial protein synthesismachinery is very similar to the prokaryotic machinery and that theaminoglycoside-induced toxicity may, at least in part, be connected todrug-mediated dysfunction of the mitochondrial ribosome.

The observed significantly increased eukaryotic anti-translationalactivity (that actually measures only the inhibition of cytoplasmicprotein synthesis and not that of mitochondrial protein synthesis)together with the significantly reduced cytotoxicity of compounds NB118,NB119, NB122, NB123, NB124, NB125, NB127 and NB128 (in comparison tothose of gentamicin and paromomycin) further support this opinion.

Example 5 Eukaryotic vs. Prokaryotic Selectivity

As discussed hereinabove, in order to constitute a worthy drug candidatewhich can be used to treat genetic diseases caused by premature stopcodon mutations, an aminoglycoside should be non-toxic and interact witheukaryotic cytoplasmic ribosomes. The virtue of non-toxicity can beverified by lack of antimicrobial activity, meaning that the drug willinhibit prokaryotic translation to a lesser extent and therefore it mostlikely will not inhibit mitochondrial translation. The presence of thisbeneficial combination of desired qualities in an aminoglycoside such asthe compounds presented herein can be demonstrated by a eukaryoticversus prokaryotic selective activity.

It can also be said that a notable selectivity of an aminoglycosidecompound towards inhibiting translation in eukaryote over inhibitingtranslation in prokaryote can be used to predict its effectiveness andsafety as a drug candidate for treating genetic disorders associatedwith premature stop codon mutations.

Table 3 consolidates and compares the results obtained for a series ofexemplary known aminoglycosides and exemplary presently disclosedaminoglycosides in translation inhibition assays conducted witheukaryotic and prokaryotic ribosomal systems. Each compound is alsonoted by the type of pharmacophores point that the compound exhibits outof the five pharmacophores points presented in Scheme 1 hereinabove. InTable 3, the pharmacophore points are denoted “i” for the hydroxyl groupin position 6′; “ii” for the AHB group in position N1; “iii” for thethird saccharide moiety “Ring III”; “iv” for a methyl at position 6′;and “v” for the methyl at position 5″.

TABLE 3 Translation Inhibition Prokaryotic versus Pharmacophore pointsIC₅₀ ^(Euk) IC₅₀ ^(Pro) Eukaryotic selectivity i ii iii iv vAminoglycoside (μM) (μM) IC₅₀ ^(Euk)/IC₅₀ ^(Pro) X X Gentamicin 62 ± 9 0.028 ± 0.004 2,214 X X Paromomycin 57 ± 4  0.051 ± 0.005 1,118 X X XG418 2.0 ± 0.3 0.009 ± 0.002 225 X X NB30 31 ± 4  0.46 ± 0.05 68 X X XNB54 24 ± 1  0.16 ± 0.02 151 X X X NB74  17 ± 0.6 1.130 ± 0.120 15 X X XX NB84 2.8 ± 0.3 0.980 ± 0.070 2.9 X X X NB118 15.5 ± 1.3  1.960 ± 0.2067.9 X X X NB119  28 ± 1.1 2.132 ± 0.478 13 X X X X NB122 5.2 ± 0.7 2.266± 0.196 2.3 X X X X NB123 4.6 ± 0.6 0.811 ± 0.059 5.7 X X X X NB124 1.49± 0.08 1.102 ± 0.185 1.3 X X X X NB125 7.96 ± 0.27 1.862 ± 0.173 4.3 X XX X X NB127 0.73 ± 0.07 1.753 ± 0.274 0.4 X X X X X NB128 0.89 ± 0.071.752 ± 0.145 0.5

In all biological experiments conducted, all tested aminoglycosides werein their sulfate salt forms. The concentrations reported in Table 3refer to that of the free amine form of each aminoglycoside. Prokaryoticand eukaryotic translation inhibition was quantified in coupledtranscription/translation assays as previously described. Thehalf-maximal concentration (IC₅₀) values were obtained from fittingconcentration response curves to the data of at least three independentexperiments, using GraFit 5 software. All the experiments were performedin duplicates and analogous results were obtained in three differentexperiments.

As can be seen in Table 3, a notable decrease in the IC₅₀ ^(Euk)/IC₅₀^(Pro) ratio (inhibition of translation in eukaryotes to inhibition oftranslation in prokaryotes) is observed, going down from an averagevalue of about 115 (average of the ratio of NB30, NB54, NB74 and NB84),to an average value of about 7 (average of the ratio of NB118, NB119,NB122 and NB123) for adding the presently disclosed pharmacophore point“v”, to an average value of about 1.6 (average of the ratio of NB124,NB125, NB127 and NB128) for adding the presently disclosed pharmacophorepoint “v” and the previously disclosed pharmacophore point “iv”.

It can clearly be seen in Table 3, that the exemplary aminoglycosidecompounds, according to some embodiments of the present invention, whichexhibit all five pharmacophores points, regardless of thestereo-configuration at the 5″ position, also exhibit the highesteukaryotic versus prokaryotic selectivity, namely these compounds areranking high in the list of possible drug candidates for treatinggenetic disorders in humans.

Indeed, while preparing and testing exemplary compounds NB124, NB125,NB127 and NB128, it has been found that the increased inhibition ofprokaryotic cytoplasmic protein synthesis is associated with increasedreadthrough activity. The data in Table 3 shows that the systematicdevelopment of a comprehensive pharmacophore could gradually increasethe specificity of the newly developed compounds to the cytoplasmicribosome and decrease their specificity to the prokaryotic ribosome,until NB127 and NB128 wherein all five pharmacophore points areimplemented, exhibit reversed selectivity to eukaryotic versusprokaryotic translation systems (ribosome).

Two observations are noted herein:

1) while the standard aminoglycoside antibiotics like gentamicin andparomomycin are 2,214-fold and 1,118-fold more selective to prokaryoticversus eukaryotic ribosome, this selectivity in G418 drops to only225-fold especially because its comparatively increased inhibition ofeukaryotic translation. This strong inhibition (IC₅₀ ^(Euk)=2 μM) ofeukaryotic translation was considered as a main reason of thedrastically high cytotoxicity of G418 as well as main reason for itsvery strong readthrough activity. The results presented in Table 3suggest that while the elevated inhibition of eukaryotic translation isindeed supports to its strong readthrough activity, the inhibition ofeukaryotic translation is not the only toxic event of G418 but that theother effect(s) of G418 on eukaryotic cells are correlated to itstoxicity.

According to the data presented in Table 3, several compounds accordingto some embodiments of the present invention, exhibit similar or greaterinhibition potency of eukaryotic translation, including NB124, NB127 andNB128, while being significantly less cytotoxic than G418.

2) plotting the IC₅₀ ^(Euk) values against the in vitro readthroughactivity of all the standard and synthetic aminoglycosides tested, closecorrelation between these two parameters has been observed, namely,increased inhibition is associated with increased readthrough activity,as illustrated in FIGS. 13A-B).

FIGS. 13A-B present scatter plots for identifying possible correlationbetween readthrough activity and protein translation inhibition in vitroin eukaryotic systems as observed for a series of known compounds andexemplary compounds according to some embodiments of the presentinvention, wherein increasing inhibition of protein synthesis (lowerIC₅₀ values) is associated with the increase of readthrough activity,whereas FIG. 13A is a sem ilogarithmic plot of eukaryotic inhibition oftranslation versus in vitro readthrough activity at 1.4 μM concentrationof the tested aminoglycosides (shown on the X-axis) using six differentnonsense mutations (W1282X, Q70X, R3X, R245X, G542X and R3381X) and FIG.1B is a linear plot of the same data presented in FIG. 13A.

It is noted that since the readthrough activity is dose dependent and isalso affected by various factors including the identity of stop codon,fourth base in the downstream sequence from the stop and the sequencecontest around the stop codon, the data presented in FIG. 13 wascollected while using one concentration (1.4 μM) in which all thecompounds were tested and series of different constructs that represent6 different constructs of 4 different disease models. Thus, increasingspecificity and selectivity to the prokaryotic ribosome leads tosubsequent increase in desired biological activity of the compound andwith reduced toxicity.

Another observation made by the present inventors involves a previouslyreported compound, NB33, which is essentially a dimer of paromamine inwhich two paromamine moieties are connected at 3′-oxygens via methylenebridge. NB33 is highly specific to eukaryotic ribosome and inhibitsprotein synthesis by IC₅₀ ^(Euk) value of 1.1 mM, almost twice as muchas G418 (IC₅₀ ^(Euk) of 2.0 mM). However, NB33 has almost no readthroughactivity, indicating that its mechanism of inhibition is different tothat of known aminoglycosides and the compounds according to someembodiments of the present invention, that exhibit readthrough activity.Thus, it was concluded that merely increasing the inhibition potency ofaminoglycoside is not necessarily accompanied with increased readthroughactivity. Such a correlation should be considered for thoseaminoglycoside compounds that inhibit translation process with a samemechanism, namely the fidelity of proof-reading process. Indeed a recentstudy on the interaction of NB33 with human A-site rRNA oligonucleotidemodel demonstrated that NB33 binds and stabilizes the A-site in anon-decoding conformation and as such blocks the ribosome translocationstep.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention. To the extent thatsection headings are used, they should not be construed as necessarilylimiting.

What is claimed is:
 1. A method for treating a genetic disorderassociated with a premature stop codon mutation, the method comprisingadministering to a subject in need thereof a therapeutically effectiveamount of the compound:

or a pharmaceutically acceptable salt thereof.
 2. The method of claim 1,wherein the genetic disorder is selected from the group consisting ofcystic fibrosis (CF), Duchenne muscular dystrophy (DMD),ataxia-telangiectasia, Hurler syndrome, hemophilia A, hemophilia B,Usher syndrome, Tay-Sachs Becker muscular dystrophy (BMD), Congenitalmuscular dystrophy (CMD), Factor VII deficiency, Familial atrialfibrillation, Hailey-Hailey disease, McArdle disease,Mucopolysaccharidosis, Nephropathic cystinosis, Polycystic kidneydisease, Rett syndrome, Spinal muscular atrophy (SMA), X-linkednephrogenic diabetes insipidus (XNDI) and X-linked retinitis pigmentosa.3. The method of claim 1, further comprising administering to thesubject an additional agent useful in treating the genetic disorder. 4.A pharmaceutical composition being packaged in a packaging material andidentified in print, in or on said packaging material, for use in thetreatment of a genetic disorder associated with a premature stop codonmutation, the composition comprising the compound:

or a pharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier.
 5. The pharmaceutical composition of claim 4,wherein said genetic disorder is selected from the group consisting ofcystic fibrosis (CF), Duchenne muscular dystrophy (DMD),ataxia-telangiectasia, Hurler syndrome, hemophilia A, hemophilia B,Usher syndrome, Tay-Sachs Becker muscular dystrophy (BMD), Congenitalmuscular dystrophy (CMD), Factor VII deficiency, Familial atrialfibrillation, Hailey-Hailey disease, McArdle disease,Mucopolysaccharidosis, Nephropathic cystinosis, Polycystic kidneydisease, Rett syndrome, Spinal muscular atrophy (SMA), X-linkednephrogenic diabetes insipidus (XNDI) and X-linked retinitis pigmentosa.